Mutations in the KCNE1 gene encoding human mink which cause arrhythmia susceptibility thereby establishing KCNE1 as an LQT gene

ABSTRACT

The genomic structure including the sequence of the intron/exon junctions is disclosed for KVLQT1 and KCNE1 which are genes associated with long QT syndrome. Additional sequence data for the two genes ARE also disclosed. Also disclosed are newly found mutations in KVLQT1 which result in long QT syndrome. The intron/exon junction sequence data allow for the design of primer pairs to amplify and sequence across all of the exons of the two genes. This can be used to screen persons for the presence of mutations which cause long QT syndrome. Assays can be performed to screen persons for the presence of mutations in either the DNA or proteins. The DNA and proteins may also be used in assays to screen for drugs which will be useful in treating or preventing the occurrence of long QT syndrome.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention is a divisional application of Ser. No. 09/135,020filed on Aug. 17, 1998, now U.S. Pat. 6,274,332 which is acontintiation-in-part of application Ser. No. 08/921,068 filed Aug. 29,1997 now abandoned, which is a continuation-in-part of application Ser.No. 08/739,383 filed Oct. 29, 1996 now abandoned, which is related toapplication Ser. No. 60/019,014 filed Dec. 22, 1995, and the presentinvention is also related to provisional application Ser. No. 60/094,477filed Jul. 29, 1998, all of which are incorporated herein by reference.

This application was made with Government support under Grant Nos. RO1HL48074, and P50-HL52338-02 (SCOR), funded by the National Institutes ofHealth, Bethesda, Md., The federal government may have certain rights inthis invention.

BACKGROUND OF THE INVENTION

The present invention is directed to genes and gene products associatedwith long QT syndrome (LQT) and to a process for the diagnosis of LQT.LQT is diagnosed in accordance with the present invention by analyzingthe DNA sequence of the KVLQT1 or KCNE1 gene of an individual to betested and comparing the respective DNA sequence to the known DNAsequence of a normal KVLQT1 or KCNE1 gene. Alternatively, the KVLQT1 orKCNE1 gene of an individual to be tested can be screened for mutationswhich cause LQT. Prediction of LQT will enable practitioners to preventthis disorder using existing medical therapy. This invention is furtherdirected to the discovery that the KVLQT1 and KCNE1 (also known as minK)proteins coassemble to form a cardiac I_(Ks), potassium channel. Thisknowledge can be used to coexpress these two proteins in a cell and sucha transformed cell can be used for screening for drugs which will beuseful in treating or preventing LQT. The invention is further directedto mutations in the human KCNE1 gene (which gene encodes human minkprotein) which have been discovered in families with LQT.

The publications and other materials used herein to illuminate thebackground of the invention or provide additional details respecting thepractice, are incorporated by reference, and for convenience arerespectively grouped in the appended List of References.

Cardiac arrhythmias are a common cause of morbidity and mortality,accounting for approximately 11% of all natural deaths (Kannel, 1987;Willich et al., 1987). In general, presymptomatic diagnosis andtreatment of individuals with life-threatening ventriculartachyarrhytlimias is poor, and in some cases medical management actuallyincreases the risk of arrhythmia and death (Cardiac ArrhythmiaSuppression Trial II Investigators, 1992). These factors make earlydetection of individuals at risk for cardiac arrhythmias and arrhythmiaprevention high priorities.

Both genetic and acquired factors contribute to the risk of developingcardiac arrhythmias. Long QT syndrome (LQT) is an inherited cardiacarrhythmia that causes abrupt loss of consciousness, syncope, seizuresand sudden death from ventricular tachyarrhythmias, specificallytorscide de pointes and ventricular fibrillation (Ward, 1964; Romano,1965; Schwartz et al., 1975; Moss et al., 1991). This disorder usuallyoccurs in young, otherwise healthy individuals (Ward, 1964; Romano,1965; Schwartz et al., 1975). Most LQT gene carriers manifestprolongation of the QT interval on electrocardiograms, a sign ofabnormal cardiac repolarization (Vincent et al., 1992). The clinicalfeatures of LQT result from episodic cardiac arrhythmias, specificallyrepolarization-related ventricular tachyairhythmias like torsade depointes, named for the characteristic undulating nature of theelectrocardiogram in this arrhythmia and ventricular fibrillation(Schwartz et al., 1975; Moss and McDonald, 1971). Torsade de pointes maydegenerate into ventricular fibrillation, a particularly lethalarrhythmia. Although LQT is not a common diagnosis, ventriculararrhythmias are very common; more than 300,000 United States citizensdie suddenly every year (Kannel, et al., 1987; Willich et al., 1987)and, in many cases, the underlying mechanism may be aberrant cardiacrepolarization. LQT, therefore, provides a unique opportunity to studylife-threatening cardiac arrhythmias at the molecular level.

Both inherited and acquired forms of LQT have been defined. Acquired LQTand secondary arrhythmias can result from cardiac ischemia, bradycardiaand metabolic abnormalities such as low serum potassium or calciumconcentration (Zipes, 1987). LQT can also result from treatment withcertain medications. including antibiotics, antihistamines, generalanesthetics, and, most commonly, antiarrhythmic medications (Zipes,1987). Inherited forms of LQT can result from mutations in at least fivedifferent genes. In previous studies, LQT loci were mapped to chromosome11p15.5 (KVLQT1 or LQT1) (Keating et al., 1991 a; Keating et al., 1991b), 7q35-36 (HERG or LQT2), 3p21-24 (SCN5A or LQT3) (Jianu et al.,1994). Of these, the most common cause of inherited LQT is KVLQT1. Ourdata indicate that mutations in this gene are responsible for more than50% of inherited LQT. Recently, a fourth LQT locus (LQT4) was mapped to4q25-27 (Schott et al., 1995). Also, KCNE1 (LQT5) has been associatedwith long QT syndrome (Splawvski et al., 1997b; Duggal et al., 1998).These genes encode ion channels involved in generation of the cardiacaction potential. Mutations can lead to channel dysfunction and delayedmyocellular repolarization. Because of regional heterogeneity of channelexpression with the myocardium, the aberrant cardiac repolarizationcreates a substrate for arrhythmia. KVLQT1 and KCNE1 are also expressedin the inner ear (Neyroud et al., 1997; Vetter et al., 1996). We andothers demonstrated that homozygous or compound heterozygous mutationsin each of these genes can cause deafness and the severe cardiacphenotype of the Jervell and Lange-Nielsen syndrome (Neyroud et al.,1997; Splawski et al., 1997a; Schultze-Bahr et al., 1997; Tyson et al.,1997). Loss of functional channels in the ear apparently disrupts theproduction of endolymph, leading to deafness.

Presymptomatic diagnosis of LQT is currently based on prolongation ofthe QT interval on electrocardiograms. QTc (Qr interval corrected forheart rate; Bazzett, 1920) greater than 0.44 second has traditionallyclassified an individual as affected. Most LQT patients, however, areyoung, otherwise healthy individuals, who do not haveelectrocardiograms. Moreover, genetic studies have shown that QTc isneither sensitive nor specific (Vincent et al., 1992). The spectrum ofQTc intervals for gene carriers and non-carriers overlaps, leading tomisclassifications. Non-carriers can have prolonged QTc intervals and bediagnosed as affected. Conversely, some LQT gene carriers have QTcintervals of ≦0.44 second but are still at increased risk forarrhythmia. Correct presymptomatic diagnosis is important for effective,gene-specific treatment of LQT.

Autosomal dominant and autosomal recessive forms of this disorder havebeen reported. Autosomal recessive LQT (also known as Jervell andLange-Nielsen syndrome) has been associated with congenital neuraldeafness; this form of LQT is rare (Jervell and Lange-Nielsen, 1957).Autosomal dominant LQT (Romano-Ward syndrome) is more common, and is notassociated with other phenotypic abnormalities (Romano et al., 1963;Ward, 1964). A disorder very similar to inherited LQT can also beacquired, usually as a result of phiarmacologic therapy (Schwvartz etal., 1975; Zipes, 1987).

The data have implications for the mechanism of arrhnthmias in LQT. Twohypotheses for LQT have previously been proposed (Schwartz et al.,1994). One suggyests that a predominance of left autonomic innervationcauses abnormal cardiac repolarization and artrliythmiiias. Thishypothesis is Supported by the finding that arrhythmlias can be inducedin dogs by removal of the right stellate gangalion. In addition,anecdotal evidence suggests that some LQT patients are effectivelytreated by β-adrenegic blocking agents and by left stellateganglionectomy (Schwartz et al., 1994). The second hypothesis forLQT-related arrhythmias suggests that nittations in cardiac-specific ionchannel genes, or genes that modulate cardiac ion channels, causedelayed myocellular repolarization. Delayed myocellular repolarizationcould promote reactivation of L-type calcium channels, resulting insecondary depolarizations (January and Riddle. 1989). These secondarydepolarizations are the likely cellular mechanism of torsade de pointesarrhythmiias (Suirawicz, 1989). This hypothesis is supported by theobservation that pharmacologic block of potassium channels can induce QTprolongation and repolarization-related arrhythmias in humans and animalModels (Antzelevitch and Sicouni, 1994). The discovery that one form ofLQT results from mutations in a cardiac potassium channel gene supportsthe mvyoceltular hypothesis.

In theory, mutations in a cardiac sodium channel gene could cause LQT.Voltage-gated sodium channels mediate rapid depolarization inventricular myocytes, and also conduct a small current during theplateau phase of the action potential (Attwell et al., 1979). Subtleabnormalities of sodium channel function (e.g.,., delayed sodium channelinactivation or altered voltage-dependence of channel inactivation)could delay cardiac repolarization, leading to QT prolongation andarrhythmias. In 1992, Gellens and colleagues cloned and characterized acardiac sodium channel gene, SCN5A (Gellens et al., 1992). The structureof this gene was similar to other, previously characterized sodiumchannels, encoding a large protein of 2016 amino acids. These channelproteins contain four homologous domains (DI-DIV), each of whichcontains six putative membrane spanning segments (S1-S6). SCN5A wasrecently mapped to chromosome 3p21, making it an excellent candidategene for LQT3 (George et al., 1995), and this gene was then proved to beassociated with LQT3) (Wang et al., 1995a).

In 1994. Warmke and Ganetzky identified a novel human cDNA. human ethera-go-go related gene (BERG, Warmke and Ganetzky, 1994). HERG waslocalized to human chromosome 7 by PCR analysis of a somatic cell hybridpanel (Warmke and Ganetzky, 1994) making it a candidate for LQT2. It haspredicted amino acid sequence homolooy to potassium channels. IERG wasisolated from a hippocampal cDNA library by homology to the Drosophilaether a-go-go gene (cag), which encodes a calcium-modulated potassiumchannel (Brtiggemann et al., 1993). HERG is not the human homolog ofeag, however, sharing only ˜50% amino acid sequence homology. HERG hasbeen shown to be associated with LQT2 (Curran et al., 1995).

LQT1 was found to be linked with the gene KVLQT1 (Q. Wang et al., 1996).Sixteen families with mutations in KVLQT1 were identified andcharacterized and it was shown that in all sixteen families there wascomplete linkage between LQT1 and KVLQT1. KVLQT1 was mapped tochromosome 11p15.5 making it a candidate gene for LQT1. KVLQT1 encodes aprotein with structural characteristics of potassium channels, andexpression of the gene as measured by Northern blot analysisdemonstrated that KVLQT1 is most strongly expressed in the heart. Oneintragenic deletion and ten different missense mutations which cause LQTwere identified in KVLQT1. These data define KVLQT1 as a novel cardiacpotassium channel gene and show that mutations in this gene causesusceptibility to ventricular tachyarrhythmias and sudden death.

It was known that one component of the I_(Ks) channel is minK, a 130amino acid protein with a single putative transmembrane domain (Takumiet al., 1988; Goldstein and Miller, 1991; Hausdorff et al., 1991; Takumiet al., 1991; Busch et al., 1992; Wang and Goldstein, 1995; KW Wang etal., 1996). The size and structure of this protein made it unlikely thatminK alone forms functional channels (Attali et al., 1993; Lesage etal., 1993). Evidence is presented that KVLQT1 and minK coassemble toform the cardiac I_(Ks) potassium channel. This was published bySanguinetti et al. (1996b). I_(Ks) dysfunction is a cause of cardiacarrhythmia. It was later shown that mutations in KCNE1 (which encodesminK) also can result in LQT (Splawski et al., 1997b).

SUMMARY OF THE INVENTION

The present invention teaches the genomic structure of the LQT genesKVLQT1 and KCNE1. This includes a teaching of the intron/exonboundaries. Also disclosed are additional sequence data not previouslyreported for both genes as well as mutations in KVLQT1 and KCNE1 whichare associated with LQT. Analysis of the KVLQT1 or KCNE1 gene willprovide an early diagnosis of subjects with LQT. The diagnostic methodcomprises analyzing the DNA sequence of the KVLQT1 and/or KCNE1 gene ofan individual to be tested and comparing it with the DNA sequence of thenative, non-variant gene. In a second embodiment, the KVLQT1 or KCNE1gene of an individual to be tested is screened for mutations which causeLQT. The ability to predict LQT will enable physicians to prevent thedisease with medical therapy such as beta blocking agents.

It is further demonstrated that KVLQT1 and KCNE1 (minK) coassemble toform a cardiac I_(Ks) potassium channel. I_(Ks) dysfunction is a causeof cardiac arrhythmia. The knowledge that these two proteins coassembleto form the I_(Ks) channel is useful for developing an assay to screenfor drugs which are useful in treating or preventing LQT1. Bycoexpressing both genes in a cell such as an oocvte it is possible toscreen for drugs which have an effect on the I_(Ks) channel, both in itswild-type and in its mutated forms. This knowledge is also useful forthe analysis of the KCVE1 cyene for an early diagnosis of subjects withLQT. The diagnostic methods are performed as noted above for KVLQT1and/or KCNE1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Pedigree structure for a portion of LQT kindred 1532. Affectedindividuals are shown as filled circles (females) or squares (males),unaffected individuals as empty symbols and individuals with equivocalphenotypes are stippled. Genotypes for chromosome 11 markers areindicated beneath each symbol and are shown as haplotypes. Marker order(top to bottom) is:Tel-HRAS-D11S922-TH-D11S1318-D11S454-D11S860-D11S12-Cen. The accuracy ofhaplotypes was ensured using genotypes from additional chromosome11p15.5 markers. Inferred genotypes are shown in brackets. Diseasechromosomes are indicated by boxes and recombination events areindicated with solid horizontal lines. Recombination events affectingdisease chromosomes occur in individuals: IV-22, IV-25, V-6, V-17, V-24,V-34, VI-13, VI-14 and VI-16. Recombination events occurring innon-disease chromosomes are not indicated. KVLQT1 is an SSCP conformerwithin KVLQT1 identified by primers 5 and 6, this conformer was onlyidentified in K1532 and represents a disease-associated mutation (allele2 is the mutant allele). Haplotype analyses indicate that KVLQT1 islocated between flanking markers D11S922 and D11S454.

FIG. 2. Physical map of the LQT1 region. Ideogram of chromosome 11indicates the approximate location of LQT1 (11p15.5). The location ofpolymorphic markers and some cosmids are indicated by vertical lines onthe map. Refined genetic mapping places LQT1 between TH and D11S454. Thedistance between TH and D11S454 was estimated by pulsed field gelanalyses as <700 kb. A physical map of the minimal set of overlappingYAC and P1 clones is shown. The locations of the KVLQT1 cDNA and trappedexons are indicated. Dashed lines in YACs indicate chimerism.

FIG. 3. Alignment of the S1-S6 region of KVLQT1 with Drosophilct Shakerpotassium channel. DMSHAKE1 (SHA) (Pongs et al., 1988). Identity (|) andsimilarity (:) are indicated. The 3 separate fragments of KVLQT1 are inorder: SEQ ID NO:107, SEQ ID NO:108 and SEQ ID NO: 109. The 3 separatefragments of DMSHAKE1 are in order: SEQ ID NO:110, SEQ ID NO:111 and SEQID NO:112.

FIG. 4. Northern analysis indicating expression of KVLQT1 in humanheart, placenta, lung, kidney and pancreas.

FIGS. 5A-5B. Genomic organization of KVLQT1 coding and 5′ and 3′untranslated regions. Positions of the introns are indicated witharrowheads. The six putative transmembrane segments (S1 to S6) and theputative pore region (Pore) are underlined. The stop codon is denoted byan asterisk. The nucleotide sequence of FIGS. 5A-5B is SEQ ID NO: 1. Theamino acid sequence of FIGS. 5A-5B is SEQ ID NO:2.

FIG. 6. Physical map and exon organization of KVLQT1. The genomic regionof KVLQT1 encompasses approximately 400 kilobases. Physical map of theminimal contig of overlapping P1 clones and the cosmid containing exon 1is shown. The location of KVLQT1 exons relative to genomic clones isindicated. Sizes of exons and distances are not drawn to scale.

FIGS. 7A-7E. KVLQT1 and hminK coexpression in CHO cells induces acurrent nearly identical to cardiac I_(Ks). A) KVLQT1 currents recordedduring 1 sec depolarizing pulses to membrane potentials of −50 to +40mV, applied from a holding potential of −80 mV. Tail currents weremeasured at −70 mV. B) Normalized isochronal activation curves for cellstransfected with KVLQT1 (n=6; 1 sec pulses) or KVLQT1 and KCNE1 (n=7;7.5 sec pulses). C-E) Currents recorded during 7.5 sec pulses to −40,−20; −10, 0, +20 and +40 mV in cells transfected with KCNE1 (C). KVLQT1(D) or KVLQT1 and KCNE1 (E). Tail currents were measured at −70 mV in D,and at −50 mV in C and E. The amplitude of steady state KVLQT1 currentat +40 mV was 0.37±0.14 nA (n=6). In cells cotransfected with KVLQT1 andKCNE1, time-dependent current during a 7.5-s pulse to +40 mV was1.62±0.39 nA (n=7).

FIGS. 8A-8C. Expression of KVLQT1 in Xenopius oocytes. A) Currentsrecorded in an oocyte injected with 12.5 ng KVLQT1 cPNA. Pulses wereapplied in 10 mV increments from −70 to +40 mV. B) Isochronal (Is)activation curve for KVLQT1 current. The V_(½) was −14.0±0.2 mV and theslope factor was 11.2±0.2 mV (n=9). C) The relationship of E_(rev)versus log[K⁺]_(c) was fit with a linear function and had a slope of49.9±0.4 mV (n=6-7 oocytes per point). Tail currents were measured atseveral voltages after 1.6 sec prepulses to +10 mV.

FIGS. 9A-9E. Coexpression of KVLQT1 and hminK suggests the presence of aKVLQT1 homologue in Xenopus oocytes. Currents were recorded at −40, −20,0, +20 and +40 mV in oocytes injected with either 5.8 ng KVLQT1 (FIG.9A), 1 ng KCNE1 (FIG. 9B), or co-injected with both cRNAs (FIG. 9C).FIG. 9D shows current-voltage relationships measured using 2 sec pulsesfor KVLQT1, and 7.5 sec pulses for hminK, or KVLQT1 and hminK (n=20cells for each condition). For oocytes injected with 60 pg or 1 ng ofKCNE1 cRNA, I_(Ks) at +40 mV was 2.11±0.12 μA and 2.20±0.18 μA. FIG. 9Eshows normalized isochronal activation curves for oocytes injected withKCNE1 (V_(½)=2.4±0.3 mV, slope=11.4±0.3 mV; n=16) or co-injected withKVLQT1 and KCNE1 cRNA (V_(½)=6.2±0.3 mV; slope=12.3±0.2 mV; n=20).

FIG. 10. Comparison of a partial human and a partial Xenopzt. KVLQT1amino acid sequence. Vertical lines indicate identical residues. TheXenopus amino acid sequence is SEQ ID NO:113 and the human amino acidsequence is SEQ ID NO:114.

FIGS. 11A-11D. KVLQT1 missense mutations cosegregate with LQT inkindreds K1532 (FIG. 11A), K2605 (FIG. 11B), K1723 (FIG. 11C) and K1807(FIG. 11D). The results of SSCP analyses with primer pair 5-6 (K1532),primer pair 9-10 (K1723, K1807), and primer pair 11-12 (K2605) are shownbelow each pedigree. Aberrant SSCP conformers (indicated by *)cosegregate with LQT in each kindred. For K1532, only eight of the 217individuals are shown. Because aberrant SSCP conformers cosegregatingwith LQT in K161 and K162 were identical to the aberrant confornerdefined in K1807, results for these kindreds are not shown. Results ofDNA sequence analyses of the normal (left) and aberrant (right)conformers are shown below each pedigree.

FIGS. 12A-12O. KVLQT1 intragenic deletions and missense mutationsassociated with LQT in kindreds K13216 (FIG. 12A), K1777 (FIG. 12B),K20925 (FIG. 12C), K2557 (FIG. 12D), K113119(FIG. 12E), K20926 (FIG.12F), K15019(FIG. 12G), K2625 (FIG. 12H). K2673 (FIG. 12I), K3698 (FIG.12J), K19187 (FIG. 12K), K22709 (FIG. 12L), K2762 (FIG. 12M), K3401(FIG. 12N) and K2824 (FIG. 120). Affected individuals are indicated byfilled circles (females) and squares (males). Unaffected individuals areindicated with empty symbols and uncertain individuals are either grayor stippled. The results of SSCP analyses with primer pair 1-2 (K13216,K2557, K13119, K15019), primer pair 7-8 (K1777, K20926), and primer pair9-10 (K20925) are shown below each pedigree in FIGS. 12A-12G (see Table5 for primer pairs). Because aberrant SSCP conformners cosegregatingwith LQT in K2050, K163 and K164 were identical to the aberrantconformers defined in K1723 and K1807, results for these kindreds arenot shown. For FIGS. 12A-12G, results of DNA sequence analyses of thenormal (left) and aberrant (right) conformers are shown below eachpedigree and the sequences shown are on the antisense strand. For FIGS.12H-12O the aberrant SSCP conformers are indicated by an arrow.

FIGS. 13A-13C. KCNE1 mutations associated with LQT. Pedigree structurefor LQT kindreds 1789 (FIG. 13A) and 1754 (FIG. 13B). Affectedindividuals are indicated by filled circles (females) or squares(males). Unaffected individuals are indicated by open symbols. Deceasedindividuals are identified by a diagonal slash. Aberrant SSCP conformersthat cosegregate with the disease are shown below each pedigree. Acommon polymorphism (G38S) that is not related to LQT is also detectedby these primers. The effect of mutations on hminK protein sequence isindicated. FIG. 13C is a schematic representation of hminK proteinshowing the location of LQT-associated mutations.

FIGS. 14A-14B. Magnitude of I_(Ks) varies as a function of injectedKCNE1 cRNA. A) Representative current tracings elicited by 7.5 secondpulses to +40 mV following injection of oocytes with 6 no/oocyte KVLQT1and a variable amount of KCNE1 cRNA, as indicated. Note the presence ofKVLQT1 current, and the absence of I_(Ks), in the oocyte injected with0.01 ng KCNE1. B) Current amplitude following a 7.5 second pulse to +40mV was normalized to peak current obtained by injection of 1.2 ng KCNE1.Values represent mean±S.E.M. N=8 oocytes/group.

FIGS. 15A-15D. Functional effects of D76N KCNE1 mutation. A) I_(Ks) waselicited by 7.5 second pulses from a holding potential of −80 mV to testpotentials of −40 to +40 mV. Deactivating tail currents were elicited byreturning membrane potential to −50 mV. B) Isochronal current-voltagerelation of I_(Ks-WT) (n=14) and I_(Ks-D976N) (n=14), demonstratingdominant negative suppression of I_(Ks) by D76N (p<0.0001). C) Thevoltage dependence of I_(Ks-D76N) activation, using a 7.5 second testpulse, is shifted by +16 mV compared to I_(Ks-WT). Smooth curves arebest fits of normalized tail currents to a Boltzmaiui function(V_(½)=10.8±0.8 mV, slope factor=12.1±0.3 mV for I_(Ks-WT); forI_(Ks-D76N) V_(½)=25.7±1.0 mV [p<0.0001, compared to I_(Ks-WT)] slopefactor=12.0±0.2 mV; n=14). D) I_(Ks-D76N) deactivates faster thani_(Ks-WT). I_(Ks) was activated by a 5 sec pulse to +20 mV, and tailcurrents were measured at the indicated potentials. Tail currents werefit to a single exponential function. Inset shows normalizeddeactivating tail currents at −50 mV, after a voltage step to +20 mV.

FIGS. 16A-16D. Functional effects of S74L KCNE1 mutation. A) I_(Ks-WT)and I_(Ks-S74L) recorded during 7.5 second depolarizations to −40, −20,0, +20 and +40 mV. Note the faster rate of deactivating I_(Ks-S74L) tailcurrents compared to I_(Ks-WT). B) Isochronal current-voltage relationfor I_(Ks-WT) and I_(Ks-S74L) (n=15). C) Voltage dependence ofI_(Ks-S74L) activation is shifted by +19 mV relative to I_(Ks-WT).Smooth curves are best fits of normalized tail currents to a Boltzmannfunction (V_(½)=13.7±0.6 mV, slope factor=16.0±0.3 mV for I_(Ks-WT); forI_(Ks-S74L)V_(½)=33.6±0.8 mV, slope factor=13.3±mV [both p<0.0001relative to I_(Ks-WT)]). D) I_(Ks-S74L) deactivates faster thanI_(Ks-WT).

FIG. 17. Physical map and exon organization of KCNE1. The two cosmidclones spanning the entire KCNE1 transcript are shown. Cosmid 1 does notextend to the end of exon 3 and cosmid 2 does not include exons I and 2.Sizes of the exons and distances are not drawn to scale.

FIG. 18. Genomic organization of the KCNE1 coding and 5′ and 3′untranslated regions. Positions of the introns are indicated witharrowheads. Note that both introns are within the 5′-untranslatedregion. The asterisk indicates the stop codon. The nucleotide sequenceof FIG. 18 is SEQ ID NO:3. The amino acid sequence of FIG. 18 is SEQ IDNO:4.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 is human KVLQT1 cDNA.

SEQ ID NO:2 is human KVLQT1 protein.

SEQ ID NO:3 is human KCNE1 cDNA.

SEQ ID NO:4 is human KCNE1 protein.

SEQ ID NOs:5-6 are hypothetical nucleic acids used to demonstratecalculation of homology.

SEQ ID NOs:7-8 are oligonucleotides used to capture and repair humanKVLQT1 cDNA (see Example 4).

SEQ ID NOs:9-40 are the intron/exoni boundaries of human KVLQT1 (Table3).

SEQ ID NOs:41-74 are primers used to amplify KVLQT1 exons (Table 4).

SEQ ID NOs:75-86 are primers used to define KVLQT1 mutations (Table 5).

SEQ ID NOs:87-92 are primer pairs used to amplify genomic KCNE1.

SEQ ID NOs:93-94 are primers used to amplify KCNE1 cDNA.

SEQ ID NOs:95-100 are intron/exon boundaries of KCNE1 (Table 8).

SEQ ID NOs:101-106 are primers to amplify KCNE1 exons (Table 9).

SEQ ID NOs:107-109 are fragments of KVLQT1 shown in FIG. 3.

SEQ ID NOs:110-112 are fragments of DMSHAKE shown in FIG. 3.

SEQ ID NO:113 is a partial Xenopus KVLQT1 shown in FIG. 10.

SEQ ID NO:114 is a partial human KVLQT1 shown in FIG. 10.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the determination of the genomicstructure of KVLQT1 and KCNE1 and to molecular variants of these geneswhich cause or are involved in the pathogenesis of LQT. It is alsodirected to the determination that KVLQT1 and minK coassemble to formcardiac I_(Ks) potassium channels. More specifically, the presentinvention relates to mutations in the KVLQT1 gene and also in the KCNE1gene and their use in the diagnosis of LQT. The present invention isfurther directed to methods of screening humans for the presence ofKVLQT1 and/or KCNE1 gene variants which cause LQT. Since LQT can now bedetected earlier (i.e., before symptoms appear) and more definitively,better treatment options will be available in those individualsidentified as having LQT. The present invention is also directed tomethods for screening for drugs useful in treating or preventing LQT1.

The present invention provides methods of screening the KVLQT1 and/orKCNE1 gene to identify mutations. Such methods may further comprise thestep of amplifying a portion of the KVLQT1 or KCNE1 gene, and mayfurther include a step of providing a set of polynucleotides which areprimers for amplification of said portion of the KVLQT1 or KCNE1 gene.The method is useful for identifying mutations for use in eitherdiagnosis of LQT or prognosis of LQT.

The present invention further demonstrates that KCNE1 (encoding KCNE1which is also referred to in the literature as minK) on chromosome 21 isalso involved in LQT. The minK protein and KVLQT1 coassemble to form aK⁻ channel. The present invention thus provides methods of screening theKCNE1 gene to identify mutations. Such methods may further comprise thestep of amplifying a portion of the KCNE1 gene, and may further includea step of providing a set of polynucleotides which arc primers foramplification of said portion of the KCNE1 gene. The method is usefulfor identifying mutations for use in either diagnosis of LQT orprognosis of LQT.

Finally, the present invention is directed to a method for screeningdrug candidates to identify drugs useful for treating or preventing LQT.Drug screening is performed by coexpressing mutant KVLQT1 and/or KCNE1genes in cells, such as oocytes, mammalian cells or transgenic animals,and assaying the effect of a drug candidate on the I_(Ks) channel. Theeffect is compared to the I_(Ks) channel activity of the wild-typeKVLQT1 and KCNE1 genes.

Proof that the KVLQT1 or KCNE1 gene is involved in causing LQT isobtained by finding sequences in DNA extracted from affected kindredmembers which create abnormal KVLQT1 or KCVE1 gene products or abnormallevels of the gene products. Such LQT susceptibility alleles willco-segregatc with the disease in large kindreds. They will also bepresent at a much higher frequency in non-kindred individuals with LQTthan in individuals in the general population. The key is to findmutations which are serious enough to cause obvious disruption to thenormal function of the gene product. These mutations can take a numberof forms. The most severe forms would be frame shift mutations or largedeletions which would cause the gene to code for an abnormal protein orone which would significantly alter protein expression. Less severedisruptive mutations would include small in-frame deletions andnonconservative base pair substitutions which would have a significanteffect on the protein produced, such as changes to or from a cysteineresidue, from a basic to an acidic amino acid or vice versa, from ahydrophobic to hydrophilic amino acid or vice versa, or other mutationswhich would affect secondary or tertiary protein structure. Silentmutations or those resulting in conservative amino acid substitutionswould not generally be expected to disrupt protein function.

According to the diagnostic and prognostic method of the presentinvention, alteration of the wild-type KVLQT1 or KCNE1 gene is detected.In addition, the method can be performed by detecting the wild-typeKVLQT1 or KCNE1 gene and confirming the lack of a cause of LQT as aresult of this locus. “Alteration of a wild-type gene” encompasses allforms of mutations including deletions, insertions and point mutationsin the coding and noncoding regions. Deletions mays be to the entiregene or of only a portion of the gene. Point mutations may result instop codons, frameshift mutations or amino acid substitutions. Somaticmutations are those which occur only in certain tissues and are notinherited in the Permline. Germline mutations can be found in any of abody's tissues and are inherited. Point mutational events may occur inregulatory regions, such as in the promoter of the gene, leading to lossor diminution of expression of the mRNA. Point mutations may alsoabolish proper RNA processing, leading to loss of expression of theKVLQT1 or KCNE1 gene product, or to a decrease in mRNA stability ortranslation efficiency.

Useful diaonostic techniques include, but are not limited to fluorescentin situ hybridization (FISH), direct DNA sequencing, PFGE analysis,Southern blot analysis, single stranded conformation analysis (SSCA),RNase protection assay, allele-specific oligonucleotide (ASO), dot blotanalysis and PCR-SSCP, as discussed in detail further below. Also usefulis the recently developed technique of DNA microchip technology.

The presence of LQT may be ascertained by testing any tissue of a humanfor mutations of the KVLQT1 gene or the KCNE1 gene. For example, aperson who has inherited a germline KVLQT1 or KCNE1 mutation would beprone to develop LQT. This can be determined by testing DNA from anytissue of the person's body. Most simply, blood can be drawn and DNAextracted from the cells of the blood. In addition, prenatal diagnosiscan be accomplished by testing fetal cells, placental cells or amnioticcells for mutations of the KVLQT1 or KCNE1 gene. Alteration of awild-type KVLQT1 or KCNE1 allele, whether, for example. by pointmutation or deletion, can be detected by any of the means discussedherein.

There are several methods that can be used to detect DNA sequencevariation. Direct DNA sequencing. either manual sequencing or automatedfluorescent sequencing can detect sequence variation. Another approachis the single-stranded conformation polymnorphism assay (SSCP) (Orita etal., 1989). This method does not detect all sequence changes, especiallyif the DNA fragment size is greater than 200 bp, but can be optimized todetect most DNA sequence variation. The reduced detection sensitivity isa disadvantage, but the increased throughput possible with SSCP makes itan attractive, viable alternative to direct sequencing for mutationdetection on a research basis. The fragments which have shifted mobilityon SSCP gels are then sequenced to determine the exact nature of the DNAsequence variation. Other approaches based on the detection ofmismatches between the two complementary DNA strands include clampeddenaturing gel electrophoresis (CDGE) (Sheffield et al. 1991),heteroduplex analysis (HA) (White et al., 1992) and chemical mismatchcleavage (CMC) (Grompe et al., 1989). None of the methods describedabove will detect large deletions, duplications or insertions, nor willthey detect a regulatory mutation which affects transcription ortranslation of the protein. Other methods which might detect theseclasses of mutations such as a protein truncation assay or theasymmetric assay, detect only specific types of mutations and would notdetect missense mutations. A review of currently available methods ofdetecting DNA sequence variation can be found in a recent review byGrompe (1993). Once a mutation is known, an allele specific detectionapproach such as allele specific oligonucleotide (ASO) hybridization canbe utilized to rapidly screen large numbers of other samples for thatsame mutation. Such a technique can utilize probes which are labeledwith gold nanoparticles to yield a visual color result (Elghanian etal., 1997).

A rapid preliminary analysis to detect polymorphisms in DNA sequencescan be performed by looking at a series of Southern blots of DNA cutwith one or more restriction enzymes, preferably with a large number ofrestriction enzymes. Each blot contains a series of normal individualsand a series of LQT cases. Southern blots displaying hybridizingfragments (differing in length from control DNA when probed withsequences near or including the KVLQT1 locus) indicate a possiblemutation. If restriction enzymes which produce very large restrictionfragments are used, then pulsed field gel electrophoresis (PFGE) isemployed.

Detection of point mutations may be accomplished by molecular cloning ofthe KVLQT1 or KCNE1 alleles and sequencing the alleles using techniqueswell known in the art. Also, the gene or portions of the gene may beamplified, e.g., by PCR or other amplification technique, and theamplified gene or amplified portions of the gene may be sequenced.

There are six well known methods for a more complete, yet stillindirect, test for confirming the presence of a susceptibilityallele: 1) single stranded conformation analysis (SSCP) (Orita et al.,1989); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell etal., 1990; Sheffield et al., 1989); 3) RNase protection assays(Finkelstein et al., 1990; Kinszlcr et al., 1991); 4) allele-specificoligonucleotides (ASOs) (Conner et al., 1983); 5) the use of proteinswhich recognize nuclotidec mismatches, such as the E coli mutS protein(Modrich, 1991); and 6) allele-specific PCR (Ruano and Kidd, 1989). Forallele-specific PCR, primers are used which hybridize at their 3′ endsto a particular KVLQT1 or KCNE1 mutation. If the particular mutation isnot present, an amplification product is not observed. AmplificationRefractory Mutation System (ARMS) can also be used, as disclosed inEuropean Patent Application Publication No. 0332435 and in Nexvtoni etal., 1989. Insertions and deletions of genes can also be detected bycloning, sequencing and amplification. In addition, restriction fragmentlength polymorphism (RFLP) probes for the gone or surrounding markergenes can be used to score alteration of an allele or an insertion in apolymorphic fragment. Such a method is particularly useful for screeningrelatives of an affected individual for the presence of the mutationfound in that individual. Other techniques for detecting insertions anddeletions as known in the art can be used.

In the first three methods (SSCP, DGGE and RNase protection assay), anew electrophoretic band appears. SSCP detects a band which migratesdifferentially because the sequence change causes a difference insingle-strand, intramolecular base pairing. RNase protection involvescleavage of the mutant polynucleotide into two or more smallerfragments. DGGE detects differences in migration rates of mutantsequences compared to wild-type sequences, using a denaturing gradientgel. In an allele-specific oligonucleotide assay, an oligonucleotide isdesigned which detects a specific sequence, and the assay is performedby detecting the presence or absence of a hybridization signal. In themutS assay, the protein binds only to sequences that contain anucleotide mismatch in a heteroduplex between mutant and wild-typesequences.

Mismatches, according to the present invention, are hybridized nucleicacid duplexes in which the two strands are not 100% complementary. Lackof total homology may be due to deletions, insertions, inversions orsubstitutions. Mismatch detection can be used to detect point mutationsin the gene or in its mRNA product. While these techniques are lesssensitive than sequencing, they are simpler to perform on a large numberof samples. An example of a mismatch cleavage technique is the RNaseprotection method. In the practice of the present invention, the methodinvolves the use of a labeled riboprobe which is complementary to thehuman wild-type KVLQT1 or KCNE1 gene coding sequence. The riboprobe andeither mRNA or DNA isolated from the person are annealed (hybridized)together and subsequently digested with the enzyme RNase A which is ableto detect some mismatches in a duplex RNA structure. If a mismatch isdetected by RNase A, it cleaves at the site of the mismatch. Thus, whenthe annealed RNA preparation is separated on an electrophoretic gelmatrix, if a mismatch has been detected and cleaved by RNase A, an RNAproduct will be seen which is smaller than the full length duplex RNAfor the riboprobe and the mRNA or DNA. The riboprobe need not be thefull length of the mRNA or gene but can be a segment of either. If theriboprobe comprises only a segment of the mRNA or gene, it will bedesirable to use a number of these probes to screen the whole mRNAsequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, throughenzymatic or chemical cleavage. See, e.g., Cotton et al., 1988; Shenk etal., 1975; Novack et al., 1986. Altematively, mismatches can be detectedby shifts in the electrophoretic mobility of mismatched duplexesrelative to matched duplexes. See. e.g., Cariello, 1988. With eitherriboprobes or DNA probes, the cellular mRNA or DNA which might contain amutation can be amplified using PCR (see below) before hybridization.Changes in DNA of the KVLQT1 or KCNE1 gene can also be detected usingSouthern hybridization, especially if the changes are grossrearrangements, such as deletions and insertions.

DNA sequences of the KVLQT1 or KCNE1 gene which have been amplified byuse of PCR may also be screened using allele-specific probes. Theseprobes are nucleic acid oligomers, each of which contains a region ofthe gene sequence harboring a known mutation. For example, one oligomermay be about 30 nucleotides in length, corresponding to a portion of thegene sequence. By use of a battery of such allele-specific probes, PCRamplification products can be screened to identify the presence of apreviously identified mutation in the gene. Hybridization ofallele-specific probes with amplified KVLQT1 or KCNE1 sequences can beperformed, for example, on a nylon filter. Hybridization to a particularprobe under high stringency hybridization conditions indicates thepresence of the same mutation in the tissue as in the allele-specificprobe.

The newly developed technique of nucleic acid analysis via microchiptechnology is also applicable to the present invention. In thistechnique, literally thousands of distinct oligonucleotide probes arebuilt up in an array on a silicon chip. Nucleic acid to be analyzed isfluorescently labeled and hybridized to the probes on the chip. It isalso possible to study nucleic acid-protein interactions using thesenucleic acid microchips. Using this technique one can determine thepresence of mutations or even sequence the nucleic acid being analyzedor one can measure expression levels of a gene of interest. The methodis one of parallel processing of many, even thousands, of probes at onceand can tremendously increase the rate of analysis. Several papers havebeen published which use this technique. Some of these are Hacia et al.,1996; Shoemaker et al., 1996; Chee et al., 1996; Lockhart et al., 1996;DeRisi et al., 1996; Lipshutz et al., 1995. This method has already beenused to screen people for mutations in the breast cancer gene BRCA1(Hacia et al., 1996). This new technology has been reviewed in a newsarticle in Chemical and Engineering News (Borman, 1996) and been thesubject of an editorial (Editorial, Nature Genetics, 1996). Also seeFodor (1997).

The most definitive test for mutations in a candidate locus is todirectly compare genomic KVLQT1 or KCNE1 sequences from patients withthose from a control population. Alternatively, one could sequencemessenger RNA after amplification, e.g., by PCR, thereby eliminating thenecessity of determining the exon structure of the candidate gene.

Mutations from patients falling outside the coding region of KVLQT1 orKCNE1 can be detected by examining the non-coding regions, such asintrons and regulatory sequences near or within the genes. An earlyindication that mutations in noncoding regions are important may comefrom Northern blot experiments that reveal messenger RNA molecules ofabnormal size or abundance in patients as compared to controlindividuals.

Alteration of KVLQT1 or KCNE1 mRNA expression can be detected by anytechniques known in the art. These include Northern blot analysis, PCRamplification and RNase protection. Diminished mRNA expression indicatesan alteration of the wild-type gene. Alteration of wild-type genes canalso be detected by screening for alteration of wild-type KVLQT1 orKCNE1 protein. For example, monoclonal antibodies immunoreactive withKVLQT1 or KCNE1 can be used to screen a tissue. Lack of cognate antigenwould indicate a mutation. Antibodies specific for products of mutantalleles could also be used to detect mutant gene product. Suchimmunological assays can be done in any convenient formats known in theart. These include Western blots, immunohistochemical assays and ELISAassays. Any means for detecting an altered KVLQT1 or KCNE1 protein canbe used to detect alteration of the wild-type KVLQT1 or KCNE1 gene.Functional assays, such as protein binding determinations, can be used.In addition, assays can be used which detect KVLQT1 or KCNE1 biochemicalfunction. Finding a mutant KVLQT1 or KCNE1 gene product indicatesalteration of a wild-type KVLQT1 or KCNE1 gene.

A mutant KVLQT1 or KCNE1 gene or gene product can also be, detected inother human body samples, such as serum, stool, urine and sputum. Thesame techniques discussed above for detection of mutant genes or geneproducts in tissues can be applied to other body samples. By screeningsuch body samples, a simple early diagnosis can be achieved for LQT.

The primer pairs of the present invention are useful for determinationof the nucleotide sequence of a particular KVLQT1 or KCNE1 allele usingPCR. The pairs of single-stranded DNA primers for KVLQT1 can be annealedto sequences within or surrounding the KVLQT1 gene on chromosome 11 inorder to prime amplifying DNA synthesis of the gene itself. The pairs ofsingle-stranded DNA primers for KCNE1 can be annealed to sequenceswithin or surrounding the KCNE1 gene on chromosome 21 in order to primeamplifying DNA synthesis of the gene itself. A complete set of theseprimers allows synthesis of all of the nucleotides of the gene codingsequences, i.e., the exons. The set of primers preferably allowssynthesis of both intron and exon sequences. Allele-specific primers canalso be used. Such primers anneal only to particular KVLQT1 or KCNE1mutant alleles, and thus will only amplify a product in the presence ofthe mutant allele as a template.

In order to facilitate subsequent cloning of amplified sequences,primers may have restriction enzyme site sequences appended to their 5′ends. Thus, all nucleotides of the primers are derived from KVLQT1 orKCNE1 sequence or sequences adjacent to KVLQT1 or KCNE1, except for thefew nucleotides necessary to form a restriction enzyme site. Suchenzymes and sites are well known in the art. The primers themselves canbe synthesized using techniques which are well known in the art.Generally, the primers can be made using oligonucleotide synthesizingmachines which are commercially available. Given the sequence of KVLQT1and KCNE1, design of particular primers is well within the skill of theart. The present invention adds to this by presenting data on theintron/exon boundaries thereby allowing one to design primers to amplifyand sequence all of the exonic regions completely.

The nucleic acid probes provided by the present invention are useful fora number of purposes. They can be used in Southern hybridization togenomic DNA and in the RNase protection method for detecting pointmutations already discussed above. The probes can be used to detect PCRamplification products. They may also be used to detect mismatches withthe KVLQT1 or KCNE1 gene or mRNA using other techniques.

It has been discovered that individuals with the wild-type KVLQT1 orKCNE1 gene do not have LQT. However. mutations which interfere with thefunction of the KVLQT1 or KCNE1 (gene product are involved in thepathogeniesis of LQT. Thus, the presence of an altered (or a mutant)KVLQT1 or KCNE1 gene which produces a protein having a loss of function,or altered function, directly causes LQT which increases the risk ofcardiac arrhythmias. In order to detect a KVLQT1 or KCNE1 gene mutation,a biological sample is prepared and analyzed for a difference betweenthe sequence of the allele being analyzed and the sequence of thewild-type allele. Mutant KVLQT1 or KCNE1 alleles can be initiallyidentified by any of the techniques described above. The mutant allelesare then sequenced to identify the specific mutation of the particularmutant allele. Altematively, mutant alleles can be initially identifiedby identifying mutant (altered) proteins, using conventional techniques.The mutant alleles are then sequenced to identify the specific mutationfor each allele. The mutations, especially those which lead to analtered function of the protein, are then used for the diagnostic andprognostic methods of the present invention.

It has also been discovered that the KVLQT1 protein coassembles with theminK protein. Thus, mutations in KCNE1 (which encodes minK) whichinterfere in the function of the KCNE1 gene product are involved in thepathogenesis of LQT. Thus, the presence of an altered (or a mutant)KCNE1 gene which produces a protein having a loss of function, oraltered function, directly causes LQT which increases the risk ofcardiac arrhythmias. In order to detect a KCNE1 gene mutation, abiological sample is prepared and analyzed for a difference between thesequence of the allele being analyzed and the sequence of the wild-tapeallele. Mutant KCNE1 alleles can be initially identified by any of thetechniques described above. The mutant alleles are then sequenced toidentify the specific mutation of the particular mutant (altered)proteins, using conventional techniques. The mutant alleles are thensequenced to identify the specific mutation for each allele. Themutations, especially those which lead to an altered function of theprotein, are then used for the diagnostic and prognostic methods of thepresent invention.

Definitions

The present invention employs the following definitions:

“Amplification of Polynucleotides” utilizes methods such as thepolymerase chain reaction (PCR). ligation amplification (or ligase chainreaction, LCR) and amplification methods based on the use of Q-betareplicase. Also useful are strand displacement amplification (SDA),thermophilic SDA, and nucleic acid sequence based amplification (3SR orNASBA). These methods are well known and widely practiced in the art.See, e.g., U.S. Pat. Nos. 4,683,195 and 4.683,202 and Innis et al., 1990(for PCR); Wu and Wallace, 1989 (for LCR); U.S. Pat. Nos. 5.270,184 and5,455,166 and Walker et al., 1992 (for SDA); Spargo et al., 1996 (forthernophilic SDA) and U.S. Pat. No. 5,409,818, Fahy et al., 1991 andCompton, 1991 for 3SR and NASBA. Reagents and hardware for conductingPCR are commercially available. Primers useful to amplify sequences fromthe KVLQT1 or KCNE1 region are preferably complementary to, andhybridize specifically to sequences in the KVLQT1 or KCNE1 region or inreglions that flank a target region therein. KVLQT1 or KCNE1 sequencesgenerated by amplification may be sequenced directly. Alternatively, butless desirably, the amplified sequence(s) may be cloned prior tosequence analysis. A method for the direct cloning and sequence analysisof enzymatically amplified (genomic segments has been described byScharf et al., 1986.

“Analte polynucleotide” and “analyte strand” refer to a single- ordouble-stranded polynucleotide which is suspected of containing a targetsequence, and which may be present in a variety of types of samples,including biological samples.

“Antibodies.” The present invention also provides polyclonal and/ormonoclonal antibodies and fragments thereof, and immunologic bindingequivalents thereof, which are capable of specifically binding to theKVLQT1 or KCNE1 polypeptide and fragments thereof or to polynucleotidesequences from the KVLQT1 or KCNE1 region. The term “antibody” is usedboth to refer to a homogeneous molecular entity. or a mixture such as aserum product made up of a plurality of different molecular entities.Polypeptides may be prepared synthetically in a peptide synthesizer andcoupled to a carrier molecule (e.g., keyhole limpet hemocyanin) andinjected over several months into rabbits. Rabbit sera is tested forimmunoreactivity to the KVLQT1 or KCNE1 polypeptide or fragment.Monoclonal antibodies may be made by injecting mice with the proteinpolypeptides. fusion proteins or fragments thereof. Monoclonalantibodies will be screened by ELISA and tested for specificimmunoreactivity with KVLQT1 or KCNE1 polypeptide or fragments thereof.See, Harlow and Lane, 1988. These antibodies will be useful in assays aswell as pharmaceuticals.

Once a sufficient quantity of desired polypeptide has been obtained, itmay be used for various purposes. A typical use is the production ofantibodies specific for binding. These antibodies may be eitherpolyclonal or monoclonal, and may be produced by in vitro or in vivotechniques well known in the art. For production of polyclonalantibodies, an appropriate target immune system. typically mouse orrabbit, is selected. Substantially purified antigen is presented to theimmune system in a fashion determined by methods appropriate for theanimal and by other parameters well known to immunologists. Typicalsites for injection are in footpads, intramuscularly, intraperitoneally,or intradernially. Of course, other species may be substituted for mouseor rabbit. Polyclonal antibodies are then purified using techniquesknown in the art, adjusted for the desired specificity.

An immunological response is usually assaved with an immunoassay.Normally, such immunoassay involve some purification of a source ofantigen, for example, that produced by the same cells and in the samefashion as the antigen. A variety of immunoassay methods are well knownin the art. See, e.g., Harlow and Lane, 1988, or Goding, 1986.

Monoclonal antibodies with affinities of 10⁻⁸ M⁻¹ or preferably 10⁻⁹ to10⁻¹⁰ or stronger will typically be made by standard procedures asdescribed, e.g. in Harlow and Lane, 1988 or Goding, 1986. Briefly,appropriate animals will be selected and the desired immunizationprotocol followed. After the appropriate period of time, the spleens ofsuch animals are excised and individual spleen cells fused, typically,to immortalized myeloma cells under appropriate selection conditions.Thereafter, the cells are clonally separated and the supernatants ofeach clone tested for their production of an appropriate antibodyspecific for the desired region of the antigen.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides. or alternatively, to selection of librariesof antibodies in phage or similar vectors. See Huse et al., 1989. Thepolypeptides and antibodies of the present invention may be used with orwithout modification. Frequently, polypeptides and antibodies will belabeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentagents. chemiluminescent agents, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817.837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241. Also, recombinant immunoglobulins may be produced (see U.S.Pat. No. 4,816,567).

“Binding partner” refers to a molecule capable of binding a ligandmolecule with high specificity, as for example, an antigen and anantigeni-specific antibody or an enzyme and its inhibitor. In general,the specific binding partners must bind with sufficient affinity toimmobilize the analyte copy/complementary strand duplex (in the case ofpolynucleotide hybridization) under the isolation conditions. Specificbinding partners are known in the art and include, for example, biotinand avidin or streptavidin, IgG and protein A, the numerous, knownreceptor-ligand couples, and complementary polynucleotide strands. Inthe case of complementary polynucleotide bindings partners. the partnersare normally at least about 15 bases in length, and may be at least 40bases in length. It is well recognized by those of skill in the art thatlengths shorter than 15 (e.g., 8 bases), between 15 and 40, and greaterthan 40 bases may also be used. The polynucleotides may be composed ofDNA, RNA, or synthetic nucleotide analogs. Further binding partners canbe identified using, e.g., the two-hybrid yeast screening assay asdescribed herein.

A “biological sample” refers to a sample of tissue or fluid suspected ofcontaining an analyte polynucleotide or polypeptide from an individualincluding, but not limited to. e.g., plasma, serum, spinal fluid, lymphfluid, the external sections of the skin, respiratory, intestinal, andgenitourinary tracts, tears, saliva, blood cells, tumors, organs, tissueand samples of in vitro cell culture constituents.

“Encode”. A polynucleotide is said to “encode” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

“Isolated” or “substantially pure”. An “isolated” or “substantiallypure” nucleic acid (e.g., an RNA, DNA or a mixed polymer) is one whichis substantially separated from other cellular components whichnaturally accompany a native human sequence or protein, e.g., ribosomes.polymerases, many other human genome sequences and proteins. The termembraces a nucleic acid sequence or protein which has been removed fromits naturally occurring environment, and includes recombinant or clonedDNA isolates and chemically synthesized analogs or analogs biologicallysynthesized by heterologous systems.

“KVLQT1 or KCNE1 Allele” refers, respectively, to normal alleles of theKVLQT1 or KCNE1 locus as swell as alleles of KVLQT1 or KCVE1 carryingvariations that cause LQT.

“KVLQT1 or KCNE1 Locus”, “KVLQT1 or KCNE1 Gene”, “KVLQT1 or KCNE1Nucleic Acids” or “KVLQT1 or KCNE1 Polynucleotide” each refer topolynucleotides, all of which are in the KVLQT1 or KCNE1 region.respectively, that are likely to be expressed in normal tissue, certainalleles of which result in LQT. The KVLQT1 or KCNE1 locus is intended toinclude coding sequences, intervening sequences and regulatory elementscontrolling transcription and/or translation. The KVLQT1 or KCNE1 locusis intended to include all allelic variations of the DNA sequence. Theterms “KCNE1” and “minK” may be used interchangeably.

These terms, when applied to a nucleic acid, refer to a nucleic acidwhich encodes a human KVLQT1 or KCNE1 polypeptide, fragment, hiomolog orvariant, including, e.g., protein fusions or deletions. The nucleicacids of the present invention will possess a sequence which is eitherderived from, or substantially similar to a natural KVLQT1- orKCNE1-encoding gene or one having substantial homology with a naturalKVLQT1- or KCNE1-encoding gene or a portion thereof.

The KVLQT1 or KCNE1 gene or nucleic acid includes normal alleles of theKVLQT1 or KCNE1 gene, respectively, including silent alleles having noeffect on the amino acid sequence of the KVLQT1 or KCNE1 polypeptide aswell as alleles leading to amino acid sequence variants of the KVLQT1 orKCNE1 polypeptide that do not substantially affect its function. Theseterms also include alleles having one or more mutations which adverselyaffect the function of the KVLQT1 or KCNE1 polypeptide. A mutation maybe a change in the KVLQT1 or KCNE1 nucleic acid sequence which producesa deleterious change in the amino acid sequence of the KVLQT1 or KCNE1polypeptide, resulting in partial or complete loss of KVLQT1 or KCNE1function, respectively, or may be a change in the nucleic acid sequencewhich results in the loss of effective KVLQT1 or KCNE1 expression or theproduction of aberrant forms of the KVLQT1 or KCNE1 polypeptide.

The KVLQT1 or KCNE1 nucleic acid may be that shown in SEQ ID NO:1(KVLQT1) or SEQ ID NO:3 (KCNE1) or it may be an allele as describedabove or a variant or derivative differing from that shown by a changewhich is one or more of addition, insertion, deletion and substitutionof one or more nucleotides of the sequence shown. Changes to thenucleotide sequence may result in an amino acid change at the proteinlevel, or not, as determined by the genetic code.

Thus, nucleic acid according to the present invention may include asequence different from the sequence shown in SEQ ID NOs:1 and 3 yetencode a polypeptide with the same amino acid sequence as shown in SEQID NOs:2 (KVLQT1) and 4 (KCNE1). That is, nucleic acids of the presentinvention include sequences which are degenerate as a result of thegenetic code. On the other hand, the encoded polypeptide may comprise anamino acid sequence which differs by one or more amino acid residuesfrom the amino acid sequence shown in SEQ ID NOs:2 and 4. Nucleic acidencoding a polypeptide which is an amino acid sequence variant,derivative or allele of the amino acid sequence shown in SEQ ID NOs:2and 4 is also provided by the present invention.

The KVLQT1 or KCNE1 gene, respectively, also refers to (a) any DNAsequence that (i) hybridizes to the complement of the DNA sequences thatencode the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4under highly stringent conditions (Ausubel et al., 1992) and (ii)encodes a gene product functionally equivalent to KVLQT1 or KCNE1, or(b) any DNA sequence that (i) hybridizes to the complement of the DNAsequences that encode the amino acid sequence set forth in SEQ ID NO:2or SEQ ID NO:4 under less stringent conditions, such as moderatelystringent conditions (Ausubel et al., 1992) and (ii) encodes a geneproduct functionally equivalent to KVLQT1 or KCNE1. The invention alsoincludes nucleic acid molecules that are the complements of thesequences described herein.

The polynucleotide compositions of this invention include RNA, cDNA,genomic DNA, synthetic forms, and mixed polymers, both sense andantisense strands, and may be chemically or biochemically modified ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those skilled in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithlioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

The present invention provides recombinant nucleic acids comprising allor part of the KVLQT1 or KCNE1 region. The recombinant construct may becapable of replicating autonomously in a host cell. Alternatively, therecombinant construct may become integrated into the chromosomal DNA ofthe host cell. Such a recombinant polynucleotide comprises apolynucleotide of genomic, cDNA, semi-synthetic. or synthetic originwhich, by virtue of its origin or manipulation, 1) is not associatedwith all or a portion of a polynucleotide with which it is associated innature: 2) is linked to a polynucleotide other than that to which it islinked in nature; or 3) does not occur in nature. Where nucleic acidaccording to the invention includes RNA, reference to the sequence shownshould be construed as reference to the RNA equivalent, with Usubstituted for T.

Therefore, recombinant nucleic acids comprising sequences otherwise notnaturally occurring are provided by this invention. Although thewild-type sequence may be employed, it will often be altered, e.g., bydeletion, substitution or insertion. cDNA or genomic libraries ofvarious types may be screened as natural sources of the nucleic acids ofthe present invention, or such nucleic acids may be provided byamplification of sequences resident in genomic DNA or other naturalsources, e.g., by PCR. The choice of cDNA libraries normally correspondsto a tissue source which is abundant in mRNA for the desired proteins.Phage libraries are normally preferred, but other types of libraries maybe used. Clones of a library are spread onto plates, transferred to asubstrate for screening, denatured and probed for the presence ofdesired sequences.

The DNA sequences used in this invention will usually comprise at leastabout five codons (15 nucleotides), more usually at least about 7-15codons, and most preferably, at least about 35 codons. One or moreintrons may also be present. This number of nucleotides is usually aboutthe minimal length required for a successful probe that would hybridizespecifically with a KVLQT1- or KCNE1-encoding sequence. In this context,oligomers of as low as 8 nucleotides, more generally 8-17 nucleotides,can be used for probes, especially in connection with chip technology.

Techniques for nucleic acid manipulation are described generally, forexample, in Sambrook et al., 1989 or Ausubel et al., 1992. Reagentsuseful in applying such techniques, such as restriction enzymes and thelike, are widely known in the art and commercially available from Suchvendors as New England BioLabs, Boehringer Mannheim, Amersham, Proinega,U.S. Biochemicals, New England Nuclear, and a number of other sources.The recombinant nucleic acid sequences used to produce fusion proteinsof the present invention may be derived from natural or syntheticsequences. Many natural gene sequences are obtainable from various cDNAor from genomic libraries using appropriate probes. See, GenBaiik.National Institutes of Health.

As used herein, a “portions” of the KVLQT1 or KCNE1 locus or region orallele is defined as having a minimal size of at least about eightnucleotides, or preferably about 15 nucleotides, or more preferably atleast about 25 nucleotides, and may have a minimal size of at leastabout 40 nucleotides. This definition includes all sizes in the range of8-40 nucleotides as well as greater than 40 nucleotides. Thus, thisdefinition includes nucleic acids of 8, 12, 15, 20, 25, 40, 60, 80, 100,200, 300, 400, 500 nucleotides, or nucleic acids having any number ofnucleotides within these ranges of values (e.g., 9, 10, 11, 16, 23, 30,38, 50, 72, 121, etc., nucleotides), or nucleic acids having more than500 nucleotides. The present invention includes all novel nucleic acidshaving at least 8 nucleotides derived from SEQ ID NO:1 or SEQ ID NO:3,its complement or functionally equivalent nucleic acid sequences. Thepresent invention does not include nucleic acids which exist in theprior art. That is, the present invention includes all nucleic acidshaving at least 8 nucleotides derived from SEQ ID NO:1 or SEQ ID NO:3with the proviso that it does not include nucleic acids existing in theprior art.

“KVLQT1 or KCNE1 protein” or “KVLQT1 or KCNE1 polypeptide” refers to aprotein or polypeptide encoded by the KVLQT1 or KCNE1 locus, variants orfragments thereof. The terms “KCNE1” and “minK” are usedinterchangeably. The term “polypeptide” refers to a polymer of aminoacids and its equivalent and does not refer to a specific length of theproduct; thus, peptides, oligopeptides and proteins are included withinthe definition of a polypeptide. This term also does not refer to, orexclude modifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations, and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids. etc.),polypeptides with substituted linkages as well as other modificationsknown in the art, both naturally and non-naturally occurring.Ordinarily, such polypeptides will be at least about 50% homologous tothe native KVLQT1 or KCNE1 sequence, preferably in excess of about 90%,and more preferably at least about 95% homologous. Also included areproteins encoded by DNA which hybridize under high or low stringencyconditions, to KVLQT1- or KCNE1-encoding nucleic acids and closelyrelated polypeptides or proteins retrieved by antisera to the KVLQT1 orKCNE1 protein(s).

The KVLQT1 or KCNE1 polypeptide may be that shown in SEQ ID NO:2 or SEQID NO:4 which may be in isolated and/or purified form, free orsubstantially free of material with which it is naturally associated.The polypeptide may, if produced by expression in a prokaryotic cell orproduced synthetically, lack native post-translational processing, suchas glycosylation. Alternatively, the present invention is also directedto polypeptides which are sequence variants, alleles or derixvatives ofthe KVLQT1 or KCNE1 poly peptide. Such polypeptides may have an aminoacid sequence which differs from that set forth in SEQ ID NO:2 or SEQ IDNO:4 by one or more of addition, substitution, deletion or insertion ofone or more amino acids. Preferred such polypeptides have KVLQT1 orKCNE1 function.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, such as stabilityagainst proteolytic cleavage, without the loss of other functions orproperties. Amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.Preferred substitutions arc ones which are conservative, that is, oneamino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and typicallyinclude substitutions within the following groups: glycine, alanine;valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and tyrosine,phenylalanine.

Certain amino acids may be substituted for other amino acids in aprotein structure without appreciable loss of interactive bindingcapacity with structures such as, for example, antigen-binding regionsof antibodies or binding sites on substrate molecules or binding siteson proteins interacting with the KVLQT1 or KCNE1 polypeptide. Since itis the interactive capacity and nature of a protein which defines thatprotein's biological functional activity, certain amino acidsubstitutions can be made in a protein sequence, and its underlying DNAcoding sequence, and nevertheless obtain a protein with like properties.In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydrophobic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). Altematively, thesubstitution of like amino acids can be made effectively on the basis ofhydrophilicity. The importance of hydrophilicity in conferringinteractive biological function of a protein is generally understood inthe art (U.S. Pat. No. 4,554,101). The use of the hydrophobic index orhydrophilicity in designing polypeptides is further discussed in U.S.Pat. No. 5,691,198.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acids, usually at least about 20 residues,more usually at least about 24 residues, typically at least about 28residues, and preferably more than about 35 residues.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. For instance, a promoter is operably linked to a codingsequence if the promoter affects its transcription or expression.

The term peptide mimetic or mimetic is intended to refer to a substancewhich has the essential biological activity of the KVLQT1 or KCNE1polypeptide. A peptide mimetic may be a peptide-containing molecule thatmimics elements of protein secondary structure (Johnson et al., 1993).The underlying rationale behind the use of peptide mimetics is that thepeptide backbone of proteins exists chiefly to orient amino acid sidechains in such a way as to facilitate molecular interactions, such asthose of antibody and antigen, enzyme and substrate or scaffoldingproteins. A peptide mimetic is designed to permit molecular interactionssimilar to the natural molecule. A mimetic may not be a peptide at all,but it will retain the essential biological activity of natural KVLQT1or KCNE1 polypeptide.

“Probes”. Polynucleotide polymorphisms associated with KVLQT1 or KCNE1alleles which predispose to LQT are detected by hybridization with apolynucleotide probe which forms a stable hybrid with that of the targetsequence, under stringent to moderately stringent hybridization and washconditions. If it is expected that the probes will be perfectlycomplementary to the target sequence, high stringency conditions will beused. Hybridization stringency may be lessened if some mismatching isexpected, for example. if variants are expected with the result that theprobe will not be completely complementary. Conditions are chosen whichrule out nonspecific/adventitious bindings, that is, which minimizenoise. (It should be noted that throughout this disclosure, if it issimply stated that “stringent” conditions are used that is meant to beread as “high stringency” conditions are used.) Since such indicationsidentify neutral DNA polymorphisms as well as mutations, theseindications need further analysis to demonstrate detection of a KVLQT1or KCNE1 susceptibility allele.

Probes for KVLQT1 or KCNE1 alleles may be derived from the sequences ofthe KVLQT1 or KCNE1 region, its cDNA, functionally equivalent sequences,or the complements thereof. The probes may be of any suitable length,which span all or a portion of the KVLQT1 or KCNE1 region. and whichallow specific hybridization to the region. If the target sequencecontains a sequence identical to that of the probe, the probes may beshort, e.g., in the range of about 8-30 base pairs, since the hybridwill be relatively stable under even stringent conditions. If somedegree of mismatch is expected with the probe, i.e., if it is suspectedthat the probe will hybridize to a variant region, a longer probe may beemployed which hybridizes to the target sequence with the requisitespecificity.

The probes will include an isolated polynucleotide attached to a labelor reporter molecule and may be used to isolate other polynucleotidesequences, having sequence similarity by standard methods. Fortechniques for preparing and labeling probes see, e.g., Sambrook et al.,1989 or Ausubel et al., 1992. Other similar polynucleotides may beselected by using homologous polynucleotides. Alternatively,polynucleotides encoding these or similar polypeptides may besynthesized or selected by use of the redundancy in the genetic code.Various codon substitutions may be introduced, e.g., by silent changes(thereby producing various restriction sites) or to optimize expressionfor a particular system. Mutations may be introduced to modify theproperties of the polypeptide, perhaps to change the polypeptidedegradation or turnover rate.

Probes comprising synthetic oligonucleotides or other polynucleotides ofthe present invention may be derived from naturally occurring orrecombinant single- or double-stranded polynucleotides. or be chemicallysynthesized. Probes may also be labeled by nick translation, Klenowfill-in reaction, or other methods known in the art.

Portions of the polynucleotide sequence having at least about eightnucleotides, usually at least about 15 nucleotides, and fewer than about9 kb, usually fewer than about 1.0 kb, from a polynucleotide sequenceencoding KVLQT1 or KCNE1 are preferred as probes. This definitiontherefore includes probes of sizes 8 nucleotides through 9000nucleotides. Thus, this definition includes probes of 8, 12, 15, 20, 25,40, 60, 80, 100, 200, 300, 400 or 500 nucleotides or probes having anynumber of nucleotides within these ranges of values (e.g., 9, 10, 11,16, 23, 30, 38, 50, 72, 121, etc., nucleotides), or probes having morethan 500 nucleotides. The probes may also be used to determine whethermRNA encoding KVLQT1 or KCNE1 is present in a cell or tissue. Thepresent invention includes all novel probes having at least 8nucleotides derived from SEQ ID NO:1 or SEQ ID NO:3, its complement orfunctionally equivalent nucleic acid sequences. The present inventiondoes not include probes which exist in the prior art. That is, thepresent invention includes all probes having at least 8 nucleotidesderived from SEQ ID NO:1 or SEQ ID NO:3 with the proviso that they donot include probes existing in the prior art.

Similar considerations and nucleotide lengths are also applicable toprimers which may be used for the amplification of all or part of theKVLQT1 or KCNE1 gene. Thus, a definition for primers includes primers of8, 12, 15, 20, 25, 40, 60, 80, 100, 200, 300, 400, 500 nucleotides, orprimers having an) number of nucleotides within these ranges of values(e.g., 9, 10, 11, 16, 23, 30, 38, 50, 72, 121, etc. nucleotides), orprimers having more than 500 nucleotides, or any number of nucleotidesbetween 500 and 9000. The primers may also be used to determine whethermRNA encoding KVLQT1 or KCNE1 is present in a cell or tissue. Thepresent invention includes all novel primers having at least 8nucleotides derived from the KVLQT1 or KCNE1 locus for amplifying theKVLQT1 or KCNE1 gene, its complement or functionally equivalent nucleicacid sequences. The present invention does not include primers whichexist in the prior art. That is, the present invention includes allprimers having at least 8 nucleotides with the proviso that it does notinclude primers existing in the prior art.

“Protein modifications or fragments” are provided by the presentinvention for KVLQT1 or KCNE1 polypeptides or fragments thereof whichare substantially homologous to primary structural sequence but whichinclude, e.g., in vitro or in vitro chemical and biochemicalmodifications or which incorporate unusual amino acids. Suchmodifications include, for example, acetylation, carboxylation,phosphorylation, glycosylation, ubiquitination, labeling, e.g., withradionuclides, and various enzymatic modifications, as will be readilyappreciated by those well skilled in the art. A variety of methods forlabeling polypeptides and of substituents or labels useful for suchpurposes are well known in the art, and include radioactive isotopessuch as ³²P, ligands which bind to labeled antiligands (e.g.,antibodies), fluorophores, chemiluminescent agents, enzymes, andantiligands which can serve as specific binding pair members for alabeled ligand. The choice of label depends on the sensitivity required,case of conjugation with the primer, stability requirements, andavailable instrumentation. Methods of labeling polypeptides are wellknown in the art. See Sambrook et al, 1989 or Ausubel et al., 1992.

Besides substantially full-length polypeptides the present inventionprovides for biologically active fragments of the polypeptides.Significant biological activities include ligand-binding, immunologicalactivity and other biological activities characteristic of KVLQT1 orKCNE1 polypeptides. Immunological activities include both immunogenicfunction in a target immune system, as well as sharing of immunologicalepitopes for binding, serving as either a competitor or substituteantigen for an epitope of the KVLQT1 or KCNE1 protein. As used herein,“epitope” refers to an antigenic determinant of a polypeptide. Anepitope could comprise three amino acids in a spatial conformation whichis unique to the epitope. Generally, an epitope consists of at leastfive such amino acids, and more usually consists of at least 8-10 suchamino acids. Methods of determining the spatial conformation of suchamino acids are known in the art.

For immunological purposes, tandem-repeat polypeptide segments may beused as immunogens, thereby producing highly antigenic proteins.Alternatively, such polypeptides will serve as highly efficientcompetitors for specific binding. Production of antibodies specific forKVLQT1 or KCNE1 polypeptides or fragments thereof is described below.

The present invention also provides for fusion polypeptides, comprisingKVLQT1 or KCNE1 polypeptides and fragments. Homologous polypeptides maybe fusions between two or more KVLQT1 or KCNE1 polypeptide sequences orbetween the sequences of KVLQT1 or KCNE1 and a related protein.Likewise, heterologous fusions may be constructed which would exhibit acombination of properties or activities of the derivative proteins. Forexample, ligand-binding or other domains may be “swapped” betweendifferent new fusion polypeptides or fragments. Such homologous orheterologous fusion polypeptides may display, for example, alteredstrength or specificity of binding. Fusion partners includeimmunoglobulins, bacterial β-galactosidase, trpE, protein A,β-lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha matingfactor. See Godowski et al., 1988.

Fusion proteins will typically be made by either recombinant nucleicacid methods, as described below, or may be chemically synthesized.Techniques for the synthesis of polypeptides are described, for example,in Merrifield (1963).

“Protein purification” refers to various methods for the isolation ofthe KVLQT1 or KCNE1 polypeptides from other biological material, such asfrom cells transformed with recombinant nucleic acids encoding KVLQT1 orKCNE1, and are well known in the art. For example, such polypeptides maybe purified by immunoaffinity chromatography employing, e.g., theantibodies provided by the present invention. Various methods of proteinpurification are well known in the art, and include those described inDeutscher, 1990 and Scopes, 1982.

The terms “isolated”, “substantially pure”, and “substantiallyhomogeneous” are used interchangeably to describe a protein orpolypeptide which has been separated from components which accompany itin its natural state. A monomeric protein is substantially pure when atleast about 60 to 75% of a sample exhibits a single polypeptidesequence. A substantially pure protein will typically comprise about 60to 90% W/W of a protein sample, more usually about 95%, and preferablywill be over about 99% pure. Protein purity or homogeneity may beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein sample, followed byvisualizing a single polypeptide band upon staining the gel. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art which are utilized for purification.

A KVLQT1 or KCNE1 protein is substantially free of naturally associatedcomponents when it is separated from the native contaminants whichaccompany it in its natural state. Thus, a polypeptide which ischemically synthesized or synthesized in a cellular system differentfrom the cell from which it naturally originates will be substantiallyfree from its naturally associated components. A protein may also berendered substantially free of naturally associated components byisolation, using protein purification techniques well known in the art.

A polypeptide produced as an expression product of an isolated andmanipulated genetic sequence is an “isolated polypeptide”, as usedherein, even if expressed in a homologous cell type. Synthetically madeforms or molecules expressed by heterologous cells are inherentlyisolated molecules.

“Recombinant nucleic acid” is a nucleic acid which is not naturallyoccurring, or which is made by the artificial combination of twootherwise separated segments of sequence. This artificial combination isoften accomplished by either chemical synthesis means, or by theartificial manipulation of isolated segments of nucleic acids, e.g., bygenetic engineering techniques. Such is usually done to replace a codonwith a redundant codon encoding the same or a conservative amino acid,while typically introducing or removing a sequence recognition site.Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a desired combination of functions.

“Regulatory sequences” refers to those sequences normally within 100 kbof the coding region of a locus, but they may also be more distant fromthe coding region, which affect the expression of the gene (includingtranscription of the gene, and translation, splicing, stability or thelike of the messenger RNA).

“Substantial homology or similarity”. A nucleic acid or fragment thereofis “substantially homologous” (“or substantially similar”) to anotherif, when optimally aligned (with appropriate nucleotide insertions ordeletions) with the other nucleic acid (or its complementary strand),there is nucleotide sequence identity in at least about 60% of thenucleotide bases, usually at least about 70%. more usually at leastabout 80%, preferably at least about 90%, and more preferably at leastabout 95-98% of the nucleotide bases.

To determine homology between two different nucleic acids, the percenthomology is to be determined using the BLASTN program “BLAST 2sequences”. This program is available for public use from the NationalCenter for Biotechnology Information (NCBI) over the Internet(http://www.ncbi.nlm.nih.gov/gorf/b12.html) (Altschul et al., 1997). Theparameters to be used are whatever combination of the following yieldsthe highest calculated percent homology (as calculated below) with thedefault parameters shown in parentheses:

Program—blastn

Matrix—0 BLOSUM62

Reward for a match—0 or 1 (1)

Penalty for a mismatch—0, −1, −2 or −3 (−2)

Open gap penalty—0, 1, 2, 3, 4 or 5 (5)

Extension gap penalty—0 or 1 (1)

Gap x_dropoff—0 or 50 (50)

Expect—10

Along with a variety of other results, this program shows a percentidentity across the complete strands or across regions of the twonucleic acids being matched. The program shows as part of the results analignment and identity of the two strands being compared. If the strandsare of equal length then the identity will be calculated across thecomplete length of the nucleic acids. If the strands are of unequallengths, then the length of the shorter nucleic acid is to be used. Ifthe nucleic acids are quite similar across a portion of their sequencesbut different across the rest of their sequences, the blastn program“BLAST 2 Sequences” will show an identity across only the similarportions, and these portions are reported individually. For purposes ofdetermining homology herein, the percent homology refers to the shorterof the two sequences being compared. If any one region is shown indifferent alignments with differing percent identities, the alignmentswhich yield the greatest homology are to be used. The averaging is to beperformed as in this example of SEQ ID NOs:5 and 6.

5′-ACCGTAGCTACGTACGTATATAGAAAGGGCGCGATCGTCGTCGCGTATGACGAC TTAGCATGC-3′(SEQ ID NO:5)

5′-ACCGGTAGCTACGACGTTATTTAGAAAGGGGTGTGTGTGTGTGTGTAAACCGGGGTTTTCGGGATCGTCCGTCGCGTATGACGACTTAGCCATGCACGGTATATCGTATTAGGACTAGCGATTGACTAG-3′ (SEQ ID NO:6)

The program “BLAST 2 Sequences” shows differing alignments of these twonucleic acids depending upon the parameters which are selected. Asexamples, four sets of parameters were selected for comparing SEQ IDNOs:5 and 6 (gap x_dropoff was 50 for all cases), with the results shownin Table 1. It is to be noted that none of the sets of parametersselected as shown in Table 1 is necessarily the best set of parametersfor comparing these sequences. The percent homology is calculated bymultiplying for each region showing identity the fraction of bases ofthe shorter strand within a region times the percent identity for thatregion and adding all of these together. For example, using the firstset of parameters shown in Table 1, SEQ ID NO:5 is the short sequence(63 bases), and two regions of identity are shown, the firstencompassing bases 4-29 (26 bases) of SEQ ID NO:5 with 92% identity toSEQ ID NO:6 and the second encompassing bases 39-59 (21 bases) of SEQ IDNO:5 with 100% identity to SEQ ID NO:6. Bases 1-3, 30-38 and 60-63 (16bases) are not shown as having any identity with SEQ ID NO:6. Percenthomology is calculated as: (26/63))(92)+(21/63)(100)+(16/63)(0)=71.3%homology. The percents of homology calculated using each of the foursets of parameters shown are listed in Table 1. Several othercombinations of parameters are possible, but they are not listed for thesake of brevity. It is seen that each set of parameters resulted in adifferent calculated percent homology. Because the result yielding thehighest percent homology is to be used, based solely on these four setsof parameters one would state that SEQ ID NOs:5 and 6 have 87.1%homology. Again it is to be noted that use of other parameters may showan even higher homology for SEQ ID NOs:5 and 6, but for brevity not allthe possible results are shown.

Alternatively, substantial homology or (similarity) exists when anucleic acid or fragment thereof will hybridize to another nucleic acid(or a complementary strand thereof) under selective hybridizationconditions, to a strand, or to its complement. Selectivity ofhybridization exists

TABLE 1 Parameter Values Open Extension Match Mismatch Gap Gap Regionsof identity (%) Homology 1 −2 5 1 4-29 of 5 and 39-59 of 5 and 71.3 5-31of 6 (92%) 71-91 of 6 (100%) 1 −2 2 1 4-29 of 5 and 33-63 of 5 and 83.75-31 of 6 (92%) 64-96 of 6 (93%) 1 −1 5 1 — 30-59 of 5 and 44.3 61-91 of6 (93%) 1 −1 2 1 4-29 of 5 and 30-63 of 5 and 87.1 5-31 of 6 (92%) 61-96of 6 (91%)

when hybridization which is substantially more selective than total lackof specificity occurs. Typically, selective hybridization will occurwhen there is at least about 55% homology over a stretch of at leastabout 14 nucleotides. preferably at least about 65%, more preferably atleast about 75%. and most preferably at least about 90%. See, Kanehisa,1984. The length of homology comparison, as described, may be overlonger stretches, and in certain embodiments will often be over astretch of at least about nine nucleotides, usually at least about 20nucleotides, more usually at least about 24 nucleotides, typically atleast about 28 nucleotides, more typically at least about 32nucleotides, and preferably at least about 36 or more nucleotides.

Nucleic acid hybridization will be affected by such conditions as saltconcentration, temperature, or organic solvents, in addition to the basecomposition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess of30° C., typically in excess of 37° C., and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1000 mM,typically less than 500 mM, and preferably less than 200 mM. However,the combination of parameters is much more important than the measure ofany single parameter. The stringency conditions are dependent on thelength of the nucleic acid and the base composition of the nucleic acidand can be determined by techniques well known in the art. See, e.g.,Wetmur and Davidson, 1968.

Probe sequences may also hybridize specifically to duplex DNA undercertain conditions to form triplex or other higher order DNA complexes.The preparation of such probes and suitable hybridization conditions arewell known in the art.

The terms “substantial homology” or “substantial identity”, whenreferring to polypeptides, indicate that the polypeptide or protein inquestion exhibits at least about 30% identity with an entirenaturally-occurring protein or a portion thereof, usually at least about70% identity, more usually at least about 80% identity, preferably atleast about 90% identity, and more preferably at least about 95%identity.

Homology, for polypeptides, is typically measured using sequenceanalysis software. See, e.g., the Sequence Analysis Software Package ofthe Genetics Computer Group, University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measures of homology assignedto various substitutions, deletions and other modifications.Conservative substitutions typically include substitutions within thefollowing groups: glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagine, glutamine, serine, threonine;lysine, arginine; and phenylalanine, tyrosine.

“Substantially similar function” refers to the function of a modifiednucleic acid or a modified protein, with reference to the wild-typeKVLQT1 or KCNE1 nucleic acid or wild-type KVLQT1 or KCNE1 polypeptide.The modified polypeptide will be substantially homologous to thewild-type KVLQT1 or KCNE1 polypeptide and will have substantially thesame function. The modified polypeptide may have an altered amino acidsequence and/or may contain modified amino acids: In addition to thesimilarity of function, the modified polypeptide may have other usefulproperties, such as a longer half-life. The similarity of function(activity) of the modified polypeptide may be substantially the same asthe activity of the wild-type KVLQT1 or KCNE1 polypeptide.Alternatively, the similarity of function (activity) of the modifiedpolypeptide may be higher than the activity of the wild-type KVLQT1 orKCNE1 polypeptide. The modified polypeptide is synthesized usingconventional techniques, or is encoded by a modified nucleic acid andproduced using conventional techniques. The modified nucleic acid isprepared by conventional techniques. A nucleic acid with a functionsubstantially similar to the wild-type KVLQT1 or KCNE1 gene functionproduces the modified protein described above.

A polypeptide “fragment”, “portion” or “segment” is a stretch of aminoacid residues of at least about five to seven contiguous amino acids,often at least about seven to nine contiguous amino acids, typically atleast about nine to 13 contiguous amino acids and, most preferably, atleast about 20 to 30 or more contiguous amino acids.

The polypeptides of the present invention, if soluble, may be coupled toa solid-phase support, e.g., nitrocellulose, nylon, column packingmaterials (e.g., Sepharose beads), magnetic beads, glass wool, plastic,metal, polymer gels, cells, or other substrates. Such supports may takethe form, for example, of beads, wells, dipsticks, or membranes.

“Target region” refers to a region of the nucleic acid which isamplified and/or detected. The term “target sequence” refers to asequence with which a probe or primer will form a stable hybrid underdesired conditions.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, and immunology. See, e.g.,Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al., 1992;Glover, 1985; Anand, 1992; Guthrie and Fink, 1991. A general discussionof techniques and materials for human gene mapping, including mapping ofhuman chromosome 1, is provided, e.g., in White and Lalouel, 1988.

Preparation of Recombinant or Chemically Synthesized Nucleic Acids;Vectors, Transformation, Host Cells

Large amounts of the polynucleotides of the present invention may beproduced by replication in a suitable host cell. Natural or syntheticpolynucleotide fragments coding for a desired fragment will beincorporated into recombinant polynucleotide constructs, usually DNAconstructs, capable of introduction into and replication in aprokaryotic or eukaryotic cell. Usually the polynucleotide constructswill be suitable for replication in a unicellular host, such as yeast orbacteria, but may also be intended for introduction to (with and withoutintegration within the genome) cultured mammalian or plant or othereukaryotic cell lines. The purification of nucleic acids produced by themethods of the present invention are described, e.g., in Sambrook etal., 1989 or Ausubel et al., 1992.

The polynucleotides of the present invention may also be produced bychemical synthesis, e.g., by the phosphoramidite method described byBeaucage and Caruthers (1981) or the triester method according toMatteucci and Caruthers (1981) and may be performed on commercial,automated oligonucleotide synthesizers. A double-stranded fragment maybe obtained from the single-stranded product of chemical synthesiseither by synthesizing the complementary strand and annealing the strandtogether under appropriate conditions or by adding the complementarystrand using DNA polymerase with an appropriate primer sequence.

Polynucleotide constructs prepared for introduction into a prokaryoticor eukaryotic host may comprise a replication system recognized by thehost, including the intended polynucleotide fragment encoding thedesired polypeptide, and will preferably also include transcription andtranslational initiation regulatory sequences operably linked to thepolypeptide encoding segment. Expression vectors may include, forexample, an origin of replication or autonomously replicating sequence(ARS) and expression control sequences, a promoter, an enhancer andnecessary processing information sites, such as ribosome-binding sites,RNA splice sites, polyadenylation sites, transcriptional terminatorsequences, and mRNA stabilizing sequences. Such vectors may be preparedby means of standard recombinant techniques well known in the art anddiscussed, for example in Sambrook et al., 1989 or Ausubel et al., 1992.

An appropriate promoter and other necessary vector sequences will beselected so as to be functional in the host, and may include, whenappropriate, those naturally associated with the KVLQT1 or KCNE1 gene.Examples of workable combinations of cell lines and expression vectorsare described in Sambrook et al., 1989 or Ausubel et al., 1992; seealso, e.g., Metzger et al., 1988. Many useful vectors are known in theart and may be obtained from such vendors as Stratagene. New EnglandBiolabs, Promega Biotech, and others. Promoters such as the trp, lac andphage promoters, tRNA promoters and glycolytic enzyme promoters may beused in prokaryotic hosts. Useful yeast promoters include promoterregions for metallothionein, 3-phosphoglycerate kinase or otherglycolytic enzymes such as enolase or glyceraldehyde-3-phosphatedehydrogenase, enzymes responsible for maltose and galactoseutilization, and others. Vectors and promoters suitable for use in yeastexpression are further described in Hitzeman et al., EP 73,675A.Appropriate non-native mammalian promoters might include the early andlate promoters from SV40 (Fiers et al., 1978) or promoters derived frommurine Molony leukemia virus, mouse tumor virus, avian sarcoma viruses,adenovirus II, bovine papilloma virus or polyoma. Insect promoters maybe derived from baculovirus. In addition, the construct may be joined toan amplifiable gene (e.g., DHFR) so that multiple copies of the gene maybe made. For appropriate enhancer and other expression controlsequences, see also Enhancers and Eukaryotic Gene Expression. ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1983). See also, e.g.,U.S. Pat. Nos. 5,691,198; 5,735,500; 5,747,469 and 5,436,146.

While such expression vectors may replicate autonomously, they may alsoreplicate by being inserted into the genome of the host cell, by methodswell known in the art.

Expression and cloning vectors will likely contain a selectable marker,a gene encoding a protein necessary for survival or growth of a hostcell transformed with the vector. The presence of this gene ensuresgrowth of only those host cells which express the inserts. Typicalselection genes encode proteins that a) confer resistance to antibioticsor other toxic substances, e.g. ampicillin, neomycin, methotrexate,etc., b) complement auxotrophic deficiencies, or c) supply criticalnutrients not available from complex media, e.g., the gene encodingD-alanine racemase for Bacilli. The choice of the proper selectablemarker will depend on the host cell, and appropriate markers fordifferent hosts are well known in the art.

The vectors containing the nucleic acids of interest can be transcribedin vitro, and the resulting RNA introduced into the host cell bywell-known methods, e.g., by injection (see, Kubo et al., 1988), or thevectors can be introduced directly into host cells by methods well knownin the art, which vary depending on the type of cellular host, includingelectroporation; transfection employing calcium chloride, rubidiumchloride calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; infection (where the vector isan infectious agent, such as a retroviral genome); and other methods.See generally, Sambrook et al., 1989 and Ausubel et al., 1992. Theintroduction of the polynucleotides into the host cell by any methodknown in the art, including, inter alia, those described above, will bereferred to herein as “transformation.” The cells into which have beenintroduced nucleic acids described above are meant to also include theprogeny of such cells.

Large quantities of the nucleic acids and polypeptides of the presentinvention may be prepared by expressing the KVLQT1 or KCNE1 nucleic acidor portions thereof in vectors or other expression vehicles incompatible prokaryotic or eukaryotic host cells. The most commonly usedprokaryotic hosts are strains of Escherichia coli, although otherprokaryotes, such as Bacillius subtilis or Pseudomonas may also be used.

Mammalian or other eukaryotic host cells, such as those of yeast,filamentous fungi, plant, insect, or amphibian or avian species, mayalso be useful for production of the proteins of the present invention.Propagation of mammalian cells in culture is per se well known. See,Jakoby and Pastan (eds.) (1979). Examples of commonly used mammalianhost cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO)cells, and WI38, BHK, and COS cell lines, although it will beappreciated by the skilled practitioner that other cell lines may beappropriate, e.g., to provide higher expression, desirable glycosylationpatterns, or other features. An example of a commonly used insect cellline is SF9.

Clones are selected by using markers depending on the mode of the vectorconstruction. The marker may be on the same or a different DNA molecule,preferably the same DNA molecule. In prokaryotic hosts, the transformantmay be selected, e.g., by resistance to ampicillin, tetracycline orother antibiotics. Production of a particular product based ontemperature sensitivity may also serve as an appropriate marker.

Prokaryotic or eukaryotic cells transformed with the polynucleotides ofthe present invention will be useful not only for the production of thenucleic acids and polypeptides of the present invention, but also, forexample, in studying the characteristics of KVLQT1 or KCNE1polypeptides.

The probes and primers based on the KVLQT1 or KCNE1 gene sequencedisclosed herein are used to identify homologous KVLQT1 or KCNE1 genesequences and proteins in other species. These gene sequences andproteins are used in the diagnostic/prognostic, therapeutic and drugscreening methods described herein for the species from which they havebeen isolated.

Methods of Use: Drug Screening

The invention is particularly useful for screening compounds by usingKVLQT1 and KCNE1 proteins in transformed cells, transfected oocytes ortransgenic animals. Since mutations in either the KVLQT1 or KCNE1protein can alter the functioning of the cardiac I_(Ks) potassiumchannel, candidate drugs are screened for effects on the channel usingcells containing either a normal KVLQT1 or KCNE1 protein and a mutantKCNE1 or KVLQT1 protein, respectively, or a mutant KVLQT1 and a mutantKCNE1 protein. The drug is added to the cells in culture or administeredto a transgenic animal and the effect on the induced current of theI_(Ks) potassium channel is compared to the induced current of a cell oranimal containing the wild-type KVLQT1 and minK. Drug candidates whichalter the induced current to a more normal level are useful for treatingor preventing LQT.

This invention is particularly useful for screening compounds by usingthe KVLQT1 or KCNE1 polypeptide or binding fragment thereof in any of avariety of drug screening techniques.

The KVLQT1 or KCNE1 polypeptide or fragment employed in such a test mayeither be free in solution, affixed to a solid support, or borne on acell surface. One method of drug screening, utilizes eucaryotic orprocaryotic host cells which are stably transformed with recombinantpolynucleotides expressing the polypeptide or fragment, preferably incompetitive binding assays. Such cells, either in viable or fixed form,can be used for standard binding assays. One may measure, for example,for the formation of complexes between a KVLQT1 or KCNE1 polypeptide orfragment and the agent being tested, or examine the degree to which theformation of a complex between a KVLQT1 or KCNE1 polypeptide or fragmentand a known ligand is interfered with by the agent being tested.

Thus, the present invention provides methods of screening for drugscomprising contacting such an agent with a KVLQT1 or KCNE1 polypeptideor fragment thereof and assaying (i) for the presence of a complexbetween the agent and the KVLQT1 or KCNE1 polypeptide or fragment, or(ii) for the presence of a complex between the KVLQT1 or KCNE1polypeptide or fragment and a ligand, by methods well known in the art.In such competitive binding assays the KVLQT1 or KCNE1 polypeptide orfragment is typically labeled. Free KVLQT1 or KCNE1 polypeptide orfragment is separated from that present in a protein:protein complex,and the amount of free (i.e., uncomplexed) label is a measure of thebinding of the agent being tested to KVLQT1 or KCNE1 or its interferencewith KVLQT1 or KCNE1 ligand binding, respectively. One may also measurethe amount of bound, rather than free, KVLQT1 or KCNE1. It is alsopossible to label the ligand rather than the KVLQT1 or KCNE1 and tomeasure the amount of ligand binding to KVLQT1 or KCNE1 in the presenceand in the absence of the drug being tested.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the KVLQT1 or KCNE1polypeptides and is described in detail in Geysen (published PCTapplication WO 84/03564). Briefly stated, large numbers of differentsmall peptide test compounds are synthesized on a solid substrate, suchas plastic pins or some other surface. The peptide test compounds arereacted with KVLQT1 or KCNE1 polypeptide and washed. Bound KVLQT1 orKCNE1 polypeptide is then detected by methods well known in the art.

Purified KVLQT1 or KCNE1 can be coated directly onto plates for use inthe aforementioned drug screening techniques. However, non-neutralizingantibodies to the polypeptide can be used to capture antibodies toimmobilize the KVLQT1 or KCNE1 polypeptide on the solid phase.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of specifically bindingthe KVLQT1 or KCNE1 polypeptide compete with a test compound for bindingto the KVLQT1 or KCNE1 polypeptide or fragments thereof. In this manner,the antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants of the KVLQT1 or KCNE1polypeptide.

The above screening methods are not limited to assays employing onlyKVLQT1 or KCNE1 but are also applicable to studying KVLQT1 - orKCNE1-protein complexes. The effect of drugs on the activity of thiscomplex is analyzed.

In accordance with these methods, the following assays are examples ofassays which can be used for screening for drug candidates.

A mutant KVLQT1 or KCNE1 hearse or as part of a fusion protein) is mixedwith a wild-type protein (per se or as part of a fusion protein) towhich wild-type KVLQT1 or KCNE1 binds. This mixing is performed in boththe presence of a drug and the absence of the drug, and the amount ofbinding of the mutant KVLQT1 or KCNE1 with the wild-type protein ismeasured. If the amount of the binding is more in the presence of saiddrug than in the absence of said drug, the drug is a drub candidate fortreating LQT resulting from a mutation in KVLQT1 or KCNE1.

A wild-type KVLQT1 or KCNE₁ (per se or as part of a fusion protein) ismixed with a wild-type protein (per se or as part of a fusion protein)to which wild-type KVLQT1 or KCNE1 binds. This mixing is performed inboth the presence of a drug and the absence of the drug, and the amountof binding of the wild-type KVLQT1 or KCNE1 with the wild-type proteinis measured. If the amount of the binding is more in the presence ofsaid drug than in the absence of said drug, the drug is a drug candidatefor treating LQT resulting from a mutation in KVLQT1 or KCNE1.

A mutant protein, which as a wild-type protein binds to KVLQT1 or KCNE1(per se or as part of a fusion protein) is mixed with a wild-type KVLQT1or KCNE1 (per se or as part of a fission protein). This mixing isperformed in both the presence of a drug and the absence of the drug,and the amount of binding of the mutant protein with the wild-typeKVLQT1 or KCNE1 is measured. If the amount of the binding is more in thepresence of said drug than in the absence of said drug, the drug is adrug candidate for treating LQT resulting from a mutation in the geneencoding the protein.

The polypeptide of the invention may also be used for screeningcompounds developed as a result of combinatorial library technology.Combinatorial library technology provides an efficient way of testing apotential vast number of different substances for ability to modulateactivity of a polypeptide. Such libraries and their use are known in theart. The use of peptide libraries is preferred. See, for example, WO97/02048.

Briefly, a method of screening for a substance which modulates activityof a polypeptide may include contacting one or more test substances withthe polypeptide in a suitable reaction medium, testing the activity ofthe treated polypeptide and comparing that activity with the activity ofthe polypeptide in comparable reaction medium untreated with the testsubstance or substances. A difference in activity between the treatedand untreated polypeptides is indicative of a modulating effect of therelevant test substance or substances.

Prior to or as well as being screened for modulation of activity, testsubstances may be screened for ability to interact with the polypeptide,e.g., in a yeast two-hybrid system (e.g., Bartel et al., 1993; Fieldsand Song, 1989; Chevray and Nathans, 1992; Lee et al., 1995). Thissystem may be used as a coarse screen prior to testing a substance foractual ability to modulate activity of the polypeptide. Alternatively,the screen could be used to screen test substances for binding to anKVLQT1 or KCNE1 specific binding partner, or to find mimetics of theKVLQT1 or KCNE1 polypeptide.

Following identification of a substance which modulates or affectspolypeptide activity, the substance may be investigated further.Furthermore, it may be manufactured and/or used in preparation, i.e.,manufacture or formulation, or a composition such as a medicament,pharmaceutical composition or drug. These may be administered toindividuals.

Thus, the present invention extends in various aspects not only to asubstance identified using a nucleic acid molecule as a modulator ofpolypeptide activity, in accordance with what is disclosed herein, butalso a pharmaceutical composition, medicament, drug or other compositioncomprising such a substance, a method comprising administration of sucha composition comprising such a substance, a method comprisingadministration of such a composition to a patient, e.g., for treatment(which may include preventative treatment) of LQT, use of such asubstance in the manufacture of a composition for administration, e.g.,for treatment of LQT, and method of making a pharmaceutical compositioncomprising admixing such a substance with a pharmaceutically acceptableexcipient, vehicle or carrier, and optionally other ingredients.

A substance identified as a modulator of polypeptide function may bepeptide or non-peptide in nature. Non-peptide “small molecules” areoften preferred for many in vivo pharmaceutical uses. Accordingly, amimetic or mimic of the substance (particularly if a peptide) may bedesigned for pharmaceutical use.

The designing of mimetics to a known pharmaceutically active compound isa known approach to the development of pharmaceuticals based on a “lead”compound. This might be desirable where the active compound is difficultor expensive to synthesize or where it is unsuitable for a particularmethod of administration, e.g., pure peptides are unsuitable activeagents for oral compositions as they tend to be quickly degraded byproteases in the alimentary canal. Mimetic design, synthesis and testingis generally used to avoid randomly screening large numbers of moleculesfor a target property.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. First, the particular parts ofthe compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,e.g., by substituting each residue in turn. Alanine scans of peptide arecommonly used to refine such peptide motifs. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its stricture is modeledaccording to its physical properties, e.g., stereochemistry, bonding,size and/or charge, using data from a range of sources, e.g.,spectroscopic techniques, x-ray diffraction data and NMR. Computationalanalysis, similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modeling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modeled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the design of themimetic.

A template molecule is then selected onto which chemical groups whichmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted onto it can conveniently be selected so that themimetic is easy to synthesize, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. Alternatively, where the mimetic ispeptide-based, further stability can be achieved by cyclizing thepeptide, increasing its rigidity. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimization ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

Methods of Use: Nucleic Acid Diagnosis and Diagnostic Kits

In order to detect the presence of a KVLQT1 or KCNE1 allele predisposingan individual to LQT, a biological sample such as blood is prepared andanalyzed for the presence or absence of susceptibility alleles of KVLQT1or KCNE1. In order to detect the presence of LQT or as a prognosticindicator, a biological sample is prepared and analyzed for the presenceor absence of mutant alleles of KVLQT1 or KCNE1. Results of these testsand interpretive information are returned to the health care providerfor communication to the tested individual. Such diagnoses may beperformed by diagnostic laboratories, or, alternatively, diagnostic kitsare manufactured and sold to health care providers or to privateindividuals for self-diagnosis.

Initially, the screening method involves amplification of the relevantKVLQT1 or KCNE1 sequences. In another preferred embodiment of theinvention, the screening method involves a non-PCR based strategy. Suchscreening methods include two-step label amplification methodologiesthat are well known in the art. Both PCR and non-PCR based screeningstrategies can detect target sequences with a high level of sensitivity.

The most popular method used today is target amplification. Here, thetarget nucleic acid sequence is amplified with polymerases. Oneparticularly preferred method using polymerase-driven amplification isthe polymerase chain reaction (PCR). The polymerase chain reaction andother polymerase-driven amplification assays can achieve over amillion-fold increase in copy number through the use ofpolymerase-driven amplification cycles. Once amplified, the resultingnucleic acid can be sequenced or used as a substrate for DNA probes.

When the probes are used to detect the presence of the target sequencesthe biological sample to be analyzed, such as blood or serum, may betreated, if desired, to extract the nucleic acids. The sample nucleicacid may be prepared in various ways to facilitate detection of thetarget sequence, e.g. denaturation, restriction digestion,electrophoresis or dot blotting. The targeted region of the analytenucleic acid usually must be at least partially single-stranded to formhybrids with the targeting sequence of the probe. If the sequence isnaturally single-stranded, denaturation will not be required. However,if the sequence is double-stranded, the sequence will probably need tobe denatured. Denaturation can be carried out by various techniquesknown in the art.

Analyte nucleic acid and probe are incubated under conditions whichpromote stable hybrid formation of the target sequence in the probe withthe putative targeted sequence in the analyte. The region of the probeswhich is used to bind to the analyte can be made completelycomplementary to the targeted region of human chromosome 11 for KVLQT1or chromosome 21 for KCNE1. Therefore, high stringency conditions aredesirable in order to prevent false positives. However, conditions ofhigh stringency are used only if the probes are complementary to regionsof the chromosome which are unique in the genome. The stringency ofhybridization is determined by a number of factors during hybridizationand during the washing procedure, including temperature, ionic strength,base composition, probe length, and concentration of formamide. Thesefactors are outlined in, for example, Maniatis et al., 1982 and Sambrooket al., 1989. Under certain circumstances, the formation of higher orderhybrids, such as triplexes, quadraplexes, etc., may be desired toprovide the means of detecting target sequences.

Detection, if any, of the resulting hybrid is usually accomplished bythe use of labeled probes. Alternatively, the probe may be unlabeled,but may be detectable by specific binding with a ligand which islabeled, either directly or indirectly. Suitable labels, and methods forlabeling probes and ligands are known in the art, and include, forexample, radioactive labels which may be incorporated by known methods(e.g., nick translation, random priming or kinasing), biotin,fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies, goldnanoparticles and the like. Variations of this basic scheme are known inthe art, and include those variations that facilitate separation of thehybrids to be detected from extraneous materials and/or that amplify thesignal from the labeled moiety. A number of these variations arereviewed in, e.g., Matthews and Kricka, 1988; Landegren et al., 1988;Mifflin, 1989; U.S. Pat. No. 4,868,105; and in EPO Publication No.225,807.

As noted above, non-PCR based screening assays are also contemplated inthis invention. This procedure hybridizes a nucleic acid probe (or ananalog such as a methyl phosphonate backbone replacing the normalphosphodiester), to the low level DNA target. This probe may have anenzyme covalently linked to the probe, such that the covalent linkagedoes not interfere with the specificity of the hybridization. Thisenzyme-probe-conjugate-target nucleic acid complex can then be isolatedaway from the free probe enzyme conjugate and a substrate is added forenzyme detection. Enzymatic activity is observed as a change in colordevelopment or luminescent output resulting in a 10³-10⁶ increase insensitivity. For an example relating to the preparation ofoligodeoxynucleotide-alkaline phosphatase conjugates and their use ashybridization probes, see Jablonski et al. (1986).

Two-step label amplification methodologies are known in the art. Theseassays work on the principle that a small ligand (such as digoxigenin,biotin, or the like) is attached to a nucleic acid probe capable ofspecifically binding KVLQT1. Allele specific probes are alsocontemplated within the scope of this example and exemplary allelespecific probes include probes encompassing the predisposing mutationsof this patent application.

In one example, the small ligand attached to the nucleic acid probe isspecifically recognized by an antibody-enzyme conjugate. In oneembodiment of this example, digoxigenin is attached to the nucleic acidprobe. Hybridization is detected by an antibody-alkaline phosphataseconjugate which turns over a chemiluminescent substrate. For methods forlabeling nucleic acid probes according to this embodiment see Martin etal., 1990. In a second example, the small ligand is recognized by asecond ligand-enzyme conjugate that is capable of specificallycomplexing to the first ligand. A well known embodiment of this exampleis tie biotin-avidin type of interactions. For methods for labelingnucleic acid probes and their use in biotin-avidin based assays seeRigby et al., 1977 and Nguyen et al., 1992.

It is also contemplated within the scope of this invention that thenucleic acid probe assays of this invention will employ a cocktail ofnucleic acid probes capable of detecting KVLQT1 or KCNE1. Thus. in oneexample to detect the presence of KVLQT1 or KCNE1 in a cell sample, morethan one probe complementary to the gene is employed and in particularthe number of different probes is alternatively two, three, or fivedifferent nucleic acid probe sequences. In another example, to detectthe presence of mutations in the KVLQT1 or KCNE1 gene sequence in apatient, more than one probe complementary to these genes is employedwhere the cocktail includes probes capable of binding to theallele-specific mutations identified in populations of patients withalterations in KVLQT1 or KCNE1. In this embodiment, any number of probescan be used, and will preferably include probes corresponding to themajor gene mutations identified as predisposing an individual to LQT.

Methods of Use: Peptide Diagnosis and Diagnostic Kits

The presence of LQT can also be detected on the basis of the alterationof wild-type KVLQT1 or KCNE1 polypeptide. Such alterations can bedetermined by sequence analysis in accordance with conventionaltechniques. More preferably, antibodies (polyclonal or monoclonal) areused to detect differences in, or the absence of KVLQT1 or KCNE1peptides. Techniques for raising and purifying antibodies are well knownin the art and any such techniques may be chosen to achieve thepreparations claimed in this invention. In a preferred embodiment of theinvention, antibodies will immunoprecipitate KVLQT1 or KCNE1 proteinsfrom solution as well as react with these proteins on Western orimmunoblots of polyacrylamide gels. In another preferred embodiment,antibodies will detect KVLQT1 or KCNE1 proteins in paraffin or frozentissue sections, using immunocytochemical techniques.

Preferred embodiments relating to methods for detecting KVLQT1 or KCNE1or their mutations include enzyme linked immunosorbent assays (ELISA),radioimmunoassays (RIA), immunoradiometric assays (IRMA) andimmunoenzymatic assays (IEMA), including sandwich assays usingmonoclonal and/or polyclonal antibodies. Exemplary sandwich assays aredescribed by David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530,hereby incorporated by reference.

Methods of Use: Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact (e.g., agonists, antagonists, inhibitors) in orderto fashion drugs which are, for example, more active or stable forms ofthe polypeptide, or which, e.g., enhance or interfere with the functionof a polypeptide in vivo. See, e.g., Hodgson, 1991. In one approach, onefirst determines the three-dimensional structure of a protein ofinterest (e.g., KVLQT1 or KCNE1 polypeptide) by x-ray crystallography,by computer modeling or most typically, by a combination of approaches.Less often, useful information regarding the structure of a polypeptidemay be gained by modeling based on the structure of homologous proteins.An example of rational drug design is the development of HIV proteaseinhibitors (Erickson et al., 1990). In addition, peptides (e.g., KVLQT1or KCNE1 polypeptide) are analyzed by an alanine scan (Wells, 1991). Inthis technique, an amino acid residue is replaced by Ala, and its effecton the peptide's activity is determined. Each of the amino acid residuesof the peptide is analyzed in this manner to determine the importantregions of the peptide.

It is also possible to isolate a target-specific antibody, selected by afunctional assay, and then to solve its crystal structure. In principle,this approach yields a pharmacore upon which subsequent drug design canbe based. It is possible to bypass protein crystallography altogether bygenerating anti-idiotypic antibodies (anti-ids) to a functional,pharmacologically active antibody. As a mirror image of a mirror image,the binding site of the anti-ids would be expected to be an analog ofthe original receptor. The anti-id could then be used to identify andisolate peptides from banks of chemically or biologically produced banksof peptides. Selected peptides would then act as the pharmacore.

Thus, one may design drugs which have, e.g., improved KVLQT1 or KCNE1polypeptide activity or stability or which act as inhibitors, agonists,antagonists, etc. of KVLQT1 or KCNE1 polypeptide activity. By virtue ofthe availability of cloned KVLQT1 or KCNE1 sequences, sufficient amountsof the KVLQT1 or KCNE1 polypeptide may be made available to perform suchanalytical studies as x-ray crystallography. In addition, the knowledgeof the KVLQT1 or KCNE1 protein sequences provided herein will guidethose employing computer modeling techniques in place of, or in additionto x-ray crystallography.

Methods of Use: Gene Therapy

According to the present invention, a method is also provided ofsupplying wild-type KVLQT1 or KCNE1 function to a cell which carries amutant KVLQT1 or KCNE1 allele, respectively. Supplying such a functionshould allow normal functioning of the recipient cells. The wild-typegene or a part of the gene may be introduced into the cell in a vectorsuch that the gene remains extrachromosomal. In such a situation, thegene will be expressed by the cell from the extrachromosomal location.More preferred is the situation where the wild-type gene or a partthereof is introduced into the mutant cell in such a way that itrecombines with the endogenous mutant gene present in the cell. Suchrecombination requires a double recombination event which results in thecorrection of the gene mutation. Vectors for introduction of genes bothfor recombination and for extrachromosomal maintenance are known in theart, and any suitable vector may be used. Methods for introducing DNAinto cells such as electroporation, calcium phosphate co-precipitationand viral transduction are known in the art, and the choice of method iswithin the competence of the practitioner.

As generally discussed above, the KVLQT1 or KCNE1 gene or fragment,where applicable, may be employed in gene therapy methods in order toincrease the amount of the expression products of such gene in cells. Itmay also be useful to increase the level of expression of a given LQTgene even in those heart cells in which the mutant gene is expressed ata “normal” level, but the gene product is not fully functional.

Gene therapy would be carried out according to generally acceptedmethods, for example, as described by Friedman (1991) or Culver (1996).Cells from a patient would be first analyzed by the diagnostic methodsdescribed above, to ascertain the production of KVLQT1 or KCNE1polypeptide in the cells. A virus or plasmid vector (see further detailsbelow), containing a copy of the KVLQT1 or KCNE1 gene linked toexpression control elements and capable of replicating inside the cells,is prepared. The vector may be capable of replicating inside the cells.Alternatively, the vector may be replication deficient and is replicatedin helper cells for use in gene therapy. Suitable vectors are known,such as disclosed in U.S. Pat. No. 5,252,479 and PCT publishedapplication WO 93/07282 and U.S. Pat. Nos. 5,691,198; 5,747,469;5,436,146 and 5,753,500. The vector is then injected into the patient.If the transfected gene is not permanently incorporated into the genomeof each of the targeted cells, the treatment may have to be repeatedperiodically.

Gene transfer systems known in the art may be useful in the practice ofthe gene therapy methods of the present invention. These include viraland nonviral transfer methods. A number of viruses have been used asgene transfer vectors or as the basis for repairing gene transfervectors, including papovaviruses (e.g., SV40, Madzak et al., 1992),adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian,1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson andAkrigg, 1992; Stratford-Perricaudet et al., 1990; Schneider et al.,1998), vaccinia virus (Moss, 1992; Moss, 1996), adeno-associated virus(Muzyczka, 1992; Ohi et al., 1990; Russell and Hirata, 1998),herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et al.,1992; Fink et al., 1992; Breakefield and Geller, 1987; Freese et al.,1990; Fink et al., 1996), lentiviruses (Naldini et al., 1996), Sindbisand Semliki Forest virus (Berglund et al., 1993), and retroviruses ofavian (Bandyopadhyay and Temin, 1984; Petropoulos et al., 1992), murine(Miller, 1992; Miller et al., 1985; Sorge et al., 1984; Mann andBaltimore, 1985; Miller et al., 1988), and human origin (Shimada et al.,1991; Helseth et al., 1990; Page et al., 1990; Buchschacher andPanganiban, 1992). Most human gene therapy protocols have been based ondisabled murine retroviruses, although adenovirus and adeno-associatedvirus are also being used.

Nonviral gene transfer methods known in the art include chemicaltechniques such as calcium phosphate coprecipitation (Graham and van derEb, 1973; Pellicer et al., 1980); mechanical techniques, for examplemicroinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster etal., 1981; Costantini and Lacy, 1981); membrane fusion-mediated transfervia liposomes (Felgner et al., 1987; Wang and Huang, 1989; Kaneda etal., 1989; Stewart et al., 1992; Nabel et al., 1990; Lim et al., 1991);and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al.,1990; Wu et al., 1991; Zenke et al., 1990; Wu et al., 1989; Wolff etal., 1991; Wagner et al., 1990; Wagner et al., 1991; Cotten et al.,1990; Curiel et al., 1992; Curiel et al., 1991). Viral-mediated genetransfer can be combined with direct in vivo gene transfer usingliposome delivery, allowing one to direct the viral vectors to the tumorcells and not into the surrounding nondividing cells. Alternatively, theretroviral vector producer cell line can be injected into tumors (Culveret al., 1992). Injection of producer cells would then provide acontinuous source of vector particles. This technique has been approvedfor use in humans with inoperable brain tumors.

In an approach which combines biological and physical gene transfermethods, plasmid DNA of any size is combined with apolylysine-conjugated antibody specific to the adenovirus hexon protein,and the resulting complex is bound to an adenovirus vector. Thetrimolecular complex is then used to infect cells. The adenovirus vectorpermits efficient binding, internalization, and degradation of theendosome before the coupled DNA is damaged. For other techniques for thedelivery of adenovirus based vectors see Schneider et al. (1998) andU.S. Pat. Nos. 5,691,198; 5,747,469; 5,436,146 and 5,753,500.

Liposome/DNA complexes have been shown to be capable of mediating directin vivo gene transfer. While in standard liposome preparations the genetransfer process is nonspecific, localized in vivo uptake and expressionhave been reported in tumor deposits, for example, following direct insitu administration (Nabel, 1992).

Expression vectors in the context of gene therapy are meant to includethose constructs containing sequences sufficient to express apolynucleotide that has been cloned therein. In viral expressionvectors, the construct contains viral sequences sufficient to supportpackaging of the construct. If the polynucleotide encodes KVLQT1 orKCNE1, expression will produce KVLQT1 or KCNE1. If the polynucleotideencodes an antisense polynucleotide or a ribozyme, expression willproduce the antisense polynucleotide or ribozyme. Thus in this context,expression does not require that a protein product be synthesized. Inaddition to the polynucleotide cloned into the expression vector, thevector also contains a promoter functional in eukaryotic cells. Thecloned polynucleotide sequence is under control of this promoter.Suitable eukaryotic promoters include those described above. Theexpression vector may also include sequences, such as selectable markersand other sequences described herein.

Gene transfer techniques which target DNA directly to heart tissue ispreferred. Receptor—mediated gene transfer, for example, is accomplishedby the conjugation of DNA (usually in the form of covalently closedsupercoiled plasmid) to a protein ligand via polylysine. Ligands arechosen on the basis of the presence of the corresponding ligandreceptors on the cell surface of the target cell/tissue type. Theseligand-DNA conjugates can be injected directly into the blood if desiredand are directed to the target tissue where receptor binding andinternalization of the DNA-protein complex occurs. To overcome theproblem of intracellular destruction of DNA, coinfection with adenoviruscan be included to disrupt endosome function.

The therapy is as follows: patients who carry a KVLQT1 or KCNE1susceptibility allele are treated with a gene delivery vehicle such thatsome or all of their heart precursor cells receive at least oneadditional copy of a functional normal KVLQT1 or KCNE1 allele. In thisstep, the treated individuals have reduced risk of LQT to the extentthat the effect of the susceptible allele has been countered by thepresence of the normal allele.

Methods of Use: Peptide Therapy

Peptides which have KVLQT1 or KCNE1 activity can be supplied to cellswhich carry a mutant or missing KVLQT1 or KCNE1 allele. Protein can beproduced by expression of the cDNA sequence in bacteria, for example,using known expression vectors. Alternatively, KVLQT1 or KCNE1polypeptide can be extracted from KVLQT1- or KCNE1-producing mammaliancells. In addition, the techniques of synthetic chemistry can beemployed to synthesize KVLQT1 or KCNE1 protein. Any of such techniquescan provide the preparation of the present invention which comprises theKVLQT1 or KCNE1 protein. The preparation is substantially free of otherhuman proteins. This is most readily accomplished by synthesis in amicroorganism or Active KVLQT1 or KCNE1 molecules can be introduced intocells by microinjection or by use of liposomes, for example.Alternatively, some active molecules may be taken up by cells; activelyor by diffusion. Supply of molecules with KVLQT1 or KCNE1 activityshould lead to partial reversal of LQT. Other molecules with KVLQT1 orKCNE1 activity (for example, peptides, drugs or organic compounds) mayalso be used to effect such a reversal. Modified polypeptides havingsubstantially similar function are also used for peptide therapy.

Methods of Use: Transformed Hosts

Animals for testing therapeutic agents can be selected after mutagenesisof whole animals or after treatment of germline cells or zygotes. Suchtreatments include insertion of mutant KVLQT1 and/or KCNE1 alleles,usually from a second animal species, as well as insertion of disruptedhomologous genes. Alternatively, the endogenous KVLQT1 or KCNE1 gene ofthe animals may be disrupted by insertion or deletion mutation or othergenetic alterations using conventional techniques (Capecchi, 1989;Valancius and Smithies, 1991; Hasty et al., 1991; Shinkai et al., 1992;Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992;Donehower et al., 1992). After test substances have been administered tothe animals, the presence of LQT must be assessed. If the test substanceprevents or suppresses the appearance of LQT, then the test substance isa candidate therapeutic agent for treatment of LQT. These animal modelsprovide an extremely important testing vehicle for potential therapeuticproducts.

Two strategies had been utilized herein to identify LQT genes, acandidate gene approach and positional cloning. Positional informationis now available for three LQT loci with LQT1 having been mapped tochromosome 11p15.5 (Keating et al., 1991a; Keating et al., 1991b), LQT2to 7q35-36 and LQT3 to 3p2-24 (Jiang et al., 1994). The presentinvention has also identified minK, on chromosome 21, as an LQT gene.The candidate gene approach relies on likely mechanistic hypothesesbased on physiology. Although little is known about the physiology ofLQT, the disorder is associated with prolongation of the QT interval onelectrocardiograms, a sign of abnormal cardiac repolarization. Thisassociation suggests that genes encoding ion channels, or theirmodulators, are reasonable candidates for LQT. This hypothesis is nowsupported by the discovery that chromosome 7-linked LQT results frommutations in HERG, a putative cardiac potassium channel gene. Aneuroendocrine calcium channel gene (CACNL1A2; Chin et al., 1991; Seinoet al., 1992) and a gene encoding a GTP-binding protein that modulatespotassium channels (GNA12, Weinstein et al., 1988; Magovcevic et al.,1992) became candidates for LQT3 based on their chromosomal location.Subsequent linkage analyses, however, have excluded these genes. It hasnow been shown that LQT3 is associated with SCN5A (Wang et al., 1995a).Despite considerable effort, however, a candidate gene approach tochromosome 11-linked LQT has not been successful. Two potassium channelgenes (KCNA4 and KCNC1) were mapped to the short arm of chromosome 11(Wymore et al., 1994), but both were excluded as candidates for LQT1 bylinkage analyses (Russell et al., 1995; the present study). All otherpreviously characterized cardiac potassium, chloride, sodium and calciumchannel genes were similarly excluded based on their chromosomallocations. The present study has used positional cloning and mutationalanalyses to identify LQT1.

The present invention has used genotypic analyses to show that KVLQT1 istightly linked to LQT1 in 16 unrelated families (details provided in theExamples). KVLQT1 is a putative cardiac potassium channel gene andcauses the chromosome 11-linked form of LQT. Genetic analyses suggestedthat KVLQT1 encodes a voltage-gated potassium channel with functionalimportance in cardiac repolarization and it is now shown that KVLQT1coassembles with KCNE1 to form a cardiac I_(Ks) potassium channel. Ifcorrect, the mechanism of chromosome 11-linked LQT probably involvesreduced repolarizing KVLQT1 current. Since potassium channels with sixtransmembrane domains are thought to be formed from homo- orhetero-tetramers (MacKinnon, 1991; MacKinnon et al., 1993; Covarrubiaset al., 1991), it is possible that LQT-associated mutations of KVLQT1act through a dominant-negative mechanism. The type and location ofKVLQT1 mutations described here are consistent with this hypothesis. Theresultant suppression of potassium channel function, in turn, wouldlikely lead to abnormal cardiac repolarization and increased risk ofventricular tachyarrhythmias. The mutations identified in HERG, and thebiophysics of potassium channel alpha subunits, suggest that chromosome7-linked LQT results from dominant-negative mutations and a resultantreduction in functional channels. In chromosome 3-linked LQT, bycontrast, the LQT-associated deletions identified in SCN5A are likely toresult in functional cardiac sodium channels with altered properties,such as delayed inactivation or altered voltage-dependence of channelinactivation. Delayed sodium channel inactivation would increase inwardsodium current, depolarizing the membrane. This effect is similar to thealtered membrane potential expected from HERG mutations where outwardpotassium current is decreased. It is unlikely that more deleteriousmutations of SCN5A would cause LQT. A reduction of the total number ofcardiac sodium channels, for example, would be expected to reduce actionpotential duration, a phenotype opposite that of LQT.

Presymptomatic diagnosis of LQT has depended on identification of QTprolongation on electrocardiograms. Unfortunately, electrocardiogramsare rarely performed in young, healthy individuals. In addition, manyLQT gene carriers have relatively normal QT intervals, and the firstsign of disease can be a fatal cardiac arrhythmia (Vincent et al.,1992). Now that more LQT genes (KVLQT1 and KCNE1) have been identifiedand have been associated with LQT, genetic testing for this disorder canbe contemplated. This will require continued mutational analyses andidentification of additional LQT genes. With more detailed phenotypicanalyses, phenotypic differences between the varied forms of LQT may bediscovered. These differences may be useful for diagnosis and treatment.

The identification of the association between the KVLQT1 and KCNE1 genemutations and LQT permits the early presymptomatic screening ofindividuals to identify those at risk for developing LQT. To identifysuch individuals, the KVLQT1 and/or KCNE1 alleles are screened formutations either directly or after cloning the alleles. The alleles aretested for the presence of nucleic acid sequence differences from thenormal allele using any suitable technique, including but not limitedto, one of the following methods: fluorescent in situ hybridization(FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis.single stranded conformation analysis (SSCP), linkage analysis, RNaseprotection assay, allele specific oligonucleotide (ASO), dot blotanalysis and PCR-SSCP analysis. Also useful is the recently developedtechnique of DNA microchip technology. For example, either (1) thenucleotide sequence of both the cloned alleles and normal KVLQT1 orKCNE1 gene or appropriate fragment (coding sequence or genomic sequence)are determined and then compared, or (2) the RNA transcripts of theKVLQT1 or KCNE1 gene or gene fragment are hybridized to single strandedwhole genomic DNA from an individual to be tested, and the resultingheteroduplex is treated with Ribonuclease A (RNase A) and run on adenaturing gel to detect the location of any mismatches. Two of thesemethods can be carried out according to the following procedures.

The alleles of the KVLQT1 or KCNE1 gene in an individual to be testedare cloned using conventional techniques. For example, a blood sample isobtained from the individual. The genomic DNA isolated from the cells inthis sample is partially digested to an average fragment size ofapproximately 20 kb. Fragments in the range from 18-21 kb are isolated.The resulting fragments are ligated into an appropriate vector. Thesequences of the clones are then determined and compared to the normalKVLQT1 or KCNE1 gene.

Alternatively, polymerase chain reactions (PCRs) are performed withprimer pairs for the 5′ region or the exons of the KVLQT1 or KCNE1 gene.PCRs can also be performed with primer pairs based on any sequence ofthe normal KVLQT1 or KCNE1 gene. For example, primer pairs for one ofthe introns can be prepared and utilized. Finally, RT-PCR can also beperformed on the mRNA. The amplified products are then analyzed bysingle stranded conformation polymorphisms (SSCP) using conventionaltechniques to identify any differences and these are then sequenced andcompared to the normal gene sequence.

Individuals can be quickly screened for common KVLQT1 or KCNE1 genevariants by amplifying the individual's DNA using suitable primer pairsand analyzing the amplified product, e.g., by dot-blot hybridizationusing allele-specific oligonucleotide probes.

The second method employs RNase A to assist in the detection ofdifferences between the normal KVLQT1 or KCNE1 gene and defective genes.This comparison is performed in steps using small (˜500 bp) restrictionfragments of the KVLQT1 or KCNE1 gene as the probe. First, the KVLQT1 orKCNE1 gene is digested with a restriction enzyme(s) that cuts the genesequence into fragments of approximately 500 bp. These fragments areseparated on an electrophoresis gel, purified from the gel and clonedindividually, in both orientations, into an SP6 vector (e.g., pSP64 orpSP65). The SP6-based plasmids containing inserts of the KVLQT1 or KCNE1gene fragments are transcribed in vitro using the SP6 transcriptionsystem, well known in the art, in the presence of [α-³²P]GTP, generatingradiolabeled RNA transcripts of both strands of the gene.

Individually, these RNA transcripts are used to form heteroduplexes withthe allelic DNA using conventional techniques. Mismatches that occur inthe RNA:DNA heteroduplex, owing to sequence differences between theKVLQT1 or KCNE1 fragment and the KVLQT1 or KCNE1 allele subclone fromthe individual, result in cleavage in the RNA strand when treated withRNase A. Such mismatches can be the result of point mutations or smalldeletions in the individual's allele. Cleavage of the RNA strand yieldstwo or more small RNA fragments, which run faster on the denaturing gelthan the RNA probe itself.

Any differences which are found, will identify an individual as having amolecular variant of the KVLQT1 or KCNE1 gene and the consequentpresence of long QT syndrome. These variants can take a number of forms.The most severe forms would be frame shift mutations or large deletionswhich would cause the gene to code for an abnormal protein or one whichwould significantly alter protein expression. Less severe disruptivemutations would include small in-frame deletions and nonconservativebase pair substitutions which would have a significant effect on theprotein produced, such as changes to or from a cysteine residue, from abasic to an acidic amino acid or vice versa, from a hydrophobic tohydrophilic amino acid or vice versa, or other mutations which wouldaffect secondary or tertiary protein structure. Silent mutations orthose resulting in conservative amino acid substitutions would notgenerally be expected to disrupt protein function.

Genetic testing will enable practitioners to identify individuals atrisk for LQT at, or even before, birth. Presymptomatic diagnosis of LQTwill enable prevention of these disorders. Existing medical therapies,including beta adrenergic blocking agents, may prevent and delay theonset of problems associated with the disease. Finally, this inventionchanges our understanding of the cause and treatment of common heartdisease like cardiac arrhythmias which account for 11% of all naturaldeaths. Existing diagnosis has focused on measuring the QT interval fromelectrocardiograms. This method is not a fully accurate indicator of thepresence of long QT syndrome. The present invention is a more accurateindicator of the presence of the disease. Genetic testing and improvedmechanistic understanding of LQT provide the opportunity for preventionof life-threatening arrhythmias through rational therapies. It ispossible, for example, that potassium channel opening agents will reducethe risk of arrhythmias in patients with KVLQT1 or KCNE1 mutations;sodium channel blocking agents, by contrast, may be a more effectivetreatment for patients with mutations that alter the function of SCN5A.Finally, these studies may provide insight into mechanisms underlyingcommon arrhythmias, as these arrhythmias are often associated withabnormal cardiac repolarization and may result from a combination ofinherited and acquired factors.

Pharmaceutical Compositions and Routes of Administration

The KVLQT1 and KCNE1 polypeptides, antibodies, peptides and nucleicacids of the present invention can be formulated in pharmaceuticalcompositions, which are prepared according to conventionalpharmaceutical compounding techniques. See, for example, Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa.). The composition may contain the active agent or pharmaceuticallyacceptable salts of the active agent. These compositions may comprise,in addition to one of the active substances, a pharmaceuticallyacceptable excipient, carrier, buffer, stabilizer or other materialswell known in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The carrier maytake a wide variety of forms depending on the form of preparationdesired for administration, e.g., intravenous, oral, intrathecal,epineural or parenteral.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, melts,powders, suspensions or emulsions. In preparing the compositions in oraldosage form, any of the usual pharmaceutical media may be employed, suchas, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, suspending agents, and the like in thecase of oral liquid preparations (such as, for example, suspensions,elixirs and solutions); or carriers such as starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations (such as, for example,powders, capsules and tablets). Because of their ease in administration,tablets and capsules represent the most advantageous oral dosage unitform, in which case solid pharmaceutical carriers are obviouslyemployed. If desired, tablets may be sugar-coated or enteric-coated bystandard techniques. The active agent can be encapsulated to make itstable to passage through the gastrointestinal tract while at the sametime allowing for passage across the blood brain barrier. See forexample, WO 96/11698.

For parenteral administration, the compound may be dissolved in apharmaceutical carrier and administered as either a solution or asuspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative or synthetic origin. The carrier may also contain otheringredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administeredintrathecally, they may also be dissolved in cerebrospinal fluid.

The active agent is preferably administered in a therapeuticallyeffective amount. The actual amount administered, and the rate andtime-course of administration, will depend on the nature and severity ofthe condition being treated. Prescription of treatment, e.g. decisionson dosage, timing, etc., is within the responsibility of generalpractitioners or specialists, and typically takes account of thedisorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners. Examples of techniques and protocols can be found inRemington's Pharmaceutical Sciences.

Alternatively, targeting therapies may be used to deliver the activeagent more specifically to certain types of cell, by the use oftargeting systems such as antibodies or cell specific ligands. Targetingmay be desirable for a variety of reasons, e.g. if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.

Instead of administering these agents directly, they could be producedin the target cell, e.g. in a viral vector such as described above or ina cell based delivery system such as described in U.S. Pat. No.5,550,050 and published PCT application Nos. WO 92/19195, WO 94/25503,WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO96/40959 and WO 97/12635, designed for implantation in a patient. Thevector could be targeted to the specific cells to be treated, or itcould contain regulatory elements which are more tissue specific to thetarget cells. The cell based delivery system is designed to be implantedin a patient's body at the desired target site and contains a codingsequence for the active agent. Alternatively, the agent could beadministered in a precursor form for conversion to the active form by anactivating agent produced in, or targeted to, the cells to be treated.See for example, EP 425,731A and WO 90/07936.

The present invention is further detailed in the following Examples,which are offered by way of illustration and are not intended to limitthe invention in any manner. Standard techniques well known in the artor the techniques specifically described below are utilized.

EXAMPLE 1 Methods for Phenotypic Evaluation

For these studies, six large LQT kindreds (K1532, K1723, K2605, K1807,K161 and K162) as well as some small kindreds and sporadic cases werestudied. LQT patients were identified from medical clinics throughoutNorth America and Europe. Two factors were considered forphenotyping: 1) historical data (the presence of syncope, the number ofsyncopal episodes, the presence of seizures, the age of onset ofsymptoms, and the occurrence of sudden death); and 2) the QT interval onelectrocardiograms corrected for heart rate (QT_(c)) (Bazzett, 1920). Toavoid misclassifying individuals, the same conservative approach tophenotypic assignment that was successful in previous studies was used(Keating et al., 1991a; Keating et al., 1991b; Jiang et al., 1994).Informed consent was obtained from each individual, or their Guardians,in accordance with local institutional review board guidelines.Phenotypic data were interpreted without knowledge of genotype.Symptomatic individuals with a corrected QT interval (QT_(c)) of 0.45seconds or greater and asymptomatic individuals with a QT_(c) of 0.47seconds or greater were classified as affected. Asymptomatic individualswith a QT_(c) of 0.41 seconds or less were classified as unaffected.Asymptomatic individuals with QT_(c) between 0.41 and 0.47 seconds andsymptomatic individuals with QT_(c) of 0.44 seconds or less wereclassified as uncertain.

EXAMPLE 2 Genotyping and Linkage Analysis

Genomic DNA was prepared from peripheral blood lymphocytes or cell linesderived from Epstein-Barr virus transformed lymphocytes using standardprocedures (Anderson and Gusella, 1984). For genotypic analyses, foursmall tandem repeat (STR) polymorphisms were used that were previouslymapped to chromosome 11p15.5: D11S922, TH, D11S1318 and D11S860 (Gyapavet al. 1994). Genotyping of RFLP markers (HRAS1, D11S454 and D11S12) wasperformed as previously described (Keating et al., 1991a).

Pairwise linkage analysis was performed using MLINK in LINKAGE v5.1(Lathrop et al., 1985). Assumed values of 0.90 for penetrance and 0.001for LQT gene frequency were used. Gene frequency was assumed to be equalbetween males and females. Male and female recombination frequencieswere considered to be equal. STR allele frequencies were 1/n wheren=number of observed alleles. Although the maximum LOD score for D11S454was identified at a recombination fraction of 0, the presence of onenon-obligate recombinant (individual VI-14, FIG. 1) places this LQT genetelomeric of D11S454.

EXAMPLE 3 Physical Mapping

Primers were designed based on sequences from TH-INS-IGFII and D11S454loci and used to identify and isolate clones from CEPH YAC librariesusing the PCR based technique (Green and Olson. 1990; Kwiatkowski etal., 1990). YAC terminal sequences were determined by inverse PCR asdescribed (Ochman et al., 1988) and used as STSs.

P1 clones were isolated using single copy probes from previouslyidentified cosmids cosQW22 (this study), cCI11-469 (D11S679), cCI11-385(D11S551), cCI11-565 (D11S601), cCI11-237 (D11S454) (Tanigami et al.,1992; Tokino et al., 1991; Sternberg, 1990). Newly isolated P1s weremapped to chromosome 11p15 by FISH or Southern analyses. End-specificriboprobes were generated from newly isolated P1s and used to identifyadditional adjacent clones (Riboprobe Gemini Core System Kit; Promega).DNA for P1 and cosmid clones was prepared using alkaline lysis plasmidisolation and purified by equilibrium centrifugation in CsCl-ethidiumbromide gradients as described (Sambrook et al., 1989). P1 insert endsequences were deternmined by cycle sequencing as described (Wang andKeating, 1994). STSs were generated based on these insert end sequences.Overlap between P1s and cosmids was calculated by summing therestriction fragments in common.

EXAMPLE 4 Isolation and Characterization of KVLQT1 Clones

An adult human cardiac cDNA library (Stratagene) was plated, and 1×10⁶plaques were screened using trapped exon 4181A as the probe. Sequencesof trapped exon 4181A were used to design oligonucleotide probes forcDNA library screening. The GENETRAPPER™ cDNA Positive Selection Systemwas used to screen 1×10¹¹ clones from a human heart cDNA library (LifeTechnologies, Inc.). The sequences of the capture and repairoligonucleotides were 5′-CAGATCCTGAGGATGCT-3′(SEQ ID NO:7) and5′-GTACCTGGCTGAGAAGG-3′(SEQ ID NO:8).

Composite cDNA sequences for KVLQT1 were obtained by end sequencing ofoverlapping cDNA clones and by primer walking. Sequencing was performedeither automatically, using Pharmacia A.L.F. automated sequencers, ormanually, using a Sequenase Version 2.0 DNA Sequencing Kit (UnitedStates Biochemical, Inc.). Database analyses and sequence analyses werecarried out using the GCG software package, IG software package, and theBLAST network service from the National Center for BiotechnologyInformation.

The partial genomic structure (from transmembrane domain S2 to S6) ofKVLQT1 was determined by cycle sequencing of P1 18B12 as described (Wangand Keating, 1994). Primers were designed based on KVLQT1 cDNA sequenceand used for cycle sequencing.

EXAMPLE 5 Mutation Analyses

SSCP was carried out as previously described (Wang et al., 1995a; Wanget al., 1995b). Normal and aberrant SSCP products were isolatedsequenced directly as described (Wang and Keating, 1994) or subclonedinto pBluescript (SK′; Stratagene) using the T-vector method (Marchuk etal., 1991). When the latter method was used, several clones weresequenced by the dideoxy chain termination method using Sequenase™Version 2.0 (United States Biochemicals, Inc.).

EXAMPLE 6 Northern Analyses

A multiple tissue Northern filter (Human MTN blot 1, Clontech) wasprobed with a ³²P-labeled KVLQT1 cDNA probe as previously described(Curran et al., 1995).

EXAMPLE 7 Refined Genetic and Physical Localization of LQT1

The precise location of LQT1 was determined by genotypic analyses inkindred 1532 (K1532), a large Utah family of northern European descent(FIG. 1). This kindred had been used in the initial study linking thefirst LQT gene, LQT1, to chromosome 11p15.5 (Keating et al., 1991a;Keating et al., 1991b). Additional family members were identified andphenotyped for a total sample size of 217 individuals. Phenotypicdetermination was performed as previously described (Keating et al.,1991a; Keating et al., 1991b; Jiang et al., 1994). Preliminary genotypicanalyses using markers at HRAS, TH, D11S454, and D11S12 included allascertained members of K1532. These experiments identified informativebranches of this family. Additional genotypic analyses were performedusing three highly polymorphic markers from chromosome 11p15.5: D11S922,D11S1318, and D11S860 (Gyapay et al., 1994). Genotypes and pairwise LODscores for each marker are shown in FIG. 1 and Table 2. Of thesemarkers, TH and D11S1318 were completely linked. Recombination wasidentified with all other markers tested, including HRAS. but in eachcase a statistically significant positive LOD score (+3 or greater) wasidentified. These data indicate that LQT1 is completely linked to TH andD11S1318 in this kindred and that the disease gene is locatedcentromeric of HRAS.

To refine localization of LQT1, haplotype analyses of K1532 wereperformed (see FIG. 1). Nine chromosomes bearing informativerecombination events were identified. Telomeric recombination eventswere observed in unaffected individual IV-22 (between D11S922 and TH),affected individual IV-25 (between D11S922 and TH), unaffectedindividual V-6 (between HRAS and D11S922), and affected individual V-24(between HRAS and D11S922). Centromeric recombination events wereidentified in unaffected individual V-17 (between D11S860 and D11S454),affected individual V-24 (between D11S860 and D11S454), unaffectedindividual V-34 (between D11S860 and D11S454), unaffected individualVI-13 (between D11S860 and D11S454), unaffected individual VI-14(between D11S454 and D11S1318), and affected

TABLE 2 Pairwise LOD Scores Between LQT1 and 11p15.5 MarkersRecombination Fraction (θ) 0.0 0.001 0.01 0.05 0.1 0.2 Z_(max)* θ_(max)†HRAS 9.67 9.94 10.50 10.38 9.62 7.57 10.59 0.021 D11S922 10.05 13.0513.85 13.59 12.59 10.01 13.92 0.019 TH 11.01 10.99 10.82 10.06 9.07 6.9611.01 0.0 D11S1318 10.30 10.29 10.13 9.40 8.47 6.50 10.30 0.0 KVLQT114.19 14.17 13.94 12.89 11.54 8.68 14.19 0.0 D11S454 11.06 11.05 10.8910.16 9.17 7.01 11.06 0.0 D11S860 5.77 6.92 8.32 9.14 8.92 7.46 9.150.058 D11S12 1.50 2.26 3.12 3.46 3.27 2.49 3.46 0.047 LOD scores werecomputed with the assumption of 90% penetrance and gene frequency of0.001 (Lathrop et al., 1985). *Z_(max)indicates maximum LOD score.†θ_(max)indicates estimated recombination fraction at Z_(max).

individual VI-16 (between D11S860 and D11S454). These data indicate thatLQT1 is located between D11S922 and D11S454. Together with recentstudies placing LQT1 centromeric of TH (Russell et al., 1995), thesedata place LQT1 in the interval between TH and D11S454.

The size of the region containing LQT1 was estimated using pulsed-fieldgel analyses with genomic probes from chromosome 11P15.5. Probes fromTH. D11S551 and D11S454 hybridized to a 700 kb Mlu I restrictionfragment (FIG. 2). These data suggested that the region containing LQT1is less than 700 kb. Physical representation of this region was achievedby screening yeast artificial chromosome (YAC) and P1 libraries withprobes from the region (Tanigami et al., 1992; Tokino et al., 1991). Theorder of these clones was confirmed using fluorescent in situhybridization (FISH) analyses as:telomere-TH-D11S551-D11S679-D11S601-D11S454-centromere. The clonesidentified in initial experiments were then used for identification ofadjacent, overlapping clones. The minimum set of clones from the LQT1interval is shown in FIG. 2.

EXAMPLE 8 Identification and Characterization of KVLQT1

Exon amplification with clones from the physical map was performed toidentify candidate genes for LQT1. Exon trapping was performed usingpSPL3B (Burn et al., 1995) on genomic P1 clones as previously described(Buckler et al., 1991; Church et al., 1994). A minimum of 128 trappedexons from each P1 clone were initially characterized by sizing the PCRproducts. From these, 400 clones were further analyzed by dideoxysequencing using an A.L.F. automated sequencer (Pharmacia). DNA sequenceand database analyses revealed eight possible exons with predicted aminoacid sequence similarity to ion channels. The highest similarity wasobtained for a 238 base pair trapped exon (4181A), with 53% similarityto potassium channel proteins from multiple species, includingsimilarity to a portion of a putative pore region. PCR analyses wereused to map 4181A to the short arm of chromosome 11 and to two P1s fromthe physical map (118A10, 18B12). These data suggested that 4181A waspart of a potassium channel gene on chromosome 11p15.5.

Two different cDNA library screening methods were used to determine iftrapped exon 4181A was part of a gene. Traditional plaque filterhybridization with an adult human cardiac cDNA library led to theidentification of a single positive clone. A variation of cDNA selectionwas used to screen a second cardiac cDNA library (the GENETRAPPER™ cDNAPositive Selection System, Life Technologies, Inc.), and twelveindependent clones were recovered. DNA sequence analyses revealedcomplete alignment with sequences derived from 4181A and the othertrapped exons described above. The longest open reading frame spanned1654 base pairs. Two consensus polyadenylation signals were identifiedupstream of the poly(A) tail in the 3′ untranslated region. The completecDNA was not obtained at this stage of the study.

The partial cDNA predicted a protein with structural characteristics ofpotassium channels. Hydropathy analyses suggested a topology of sixmajor hydrophobic regions that may represent membrane-spanningα-helices. These regions share sequence similarity with potassiumchannel transmembrane domains S1-S6. A comparison of the predicted aminoacid sequence derived from the identified gene and the Shaker (SHA)potassium channel (Pongs et al., 1988) is shown in FIG. 3. In the regioncontaining S1-S6, the amino acid sequence identity was 30% andsimilarity was 59%. The sequence located 3′ of S1-S6 did not havesignificant similarity to any known protein. Because this gene has highsimilarity to voltage-gated potassium channel genes and became a strongcandidate for LQT1, it was named KVLQT1.

Northern blot analyses were used to determine the tissue distribution ofKVLQT1 mRNA. KVLQT1 cDNA probes detected a 3.2 kb transcript in humanpancreas, heart, kidney, lung, and placenta, but not in skeletal muscle,liver, or brain (FIG. 4). The heart showed highest levels of KVLQT1mRNA. The Northern analyses were performed using a multiple tissueNorthern filter (Human MTN blot 1, Clontech) as described by Curran etal., 1995.

EXAMPLE 9 Characterization of the Complete KVLQT1 cDNA

The studies described above resulted in the cloning and characterizationof an incomplete cDNA for KVLQT1. The sequence of this incomplete cDNApredicted a protein with six hydrophobic membrane-spanning α-helices(S1-S6) and a typical K⁺ channel pore signature sequence (Heginbotham etal., 1994). However, this cDNA appeared to be missing the amino terminaldomain and did not functionally express. To define the complete sequenceof KVLQT1, several cDNA libraries were screened and a new clone wasisolated. A cDNA probe containing exons 3 through 6 was used to isolatethree full length KVLQT1 cDNA clones from an adult heart cDNA libraryprepared in the laboratory using SuperScript Choice system (GIBCO BRL).The complete cDNA sequence and the encoded protein are shown in FIGS.5A-5B.

EXAMPLE 10 Genomic Structure of KVLQT1

The genomic DNA of KVLQT1 was examined and the exon/intron boundariesdetermined for all exons.

A. Isolation of cDNA Clones

A cDNA probe containing exons 3 through 6 was used to isolate three fulllength KVLQT1 cDNA clones from an adult heart cDNA library prepared inthe laboratory using SuperScript Choice system (GIBCO BRL).

B. Isolation of Genomic Clones

KVLQT1 P1 clones were isolated as described (Wang et al., 1996). Thecosmid containing exon 1 was isolated screening a human genomic cosmidlibrary (Stratagene) with a cDNA probe from exon 1.

C. Exon/Intron Boundary Determination

All genomic clones were sequenced using primers designed to the cDNAsequences. The KVLQT1 P1 clones were cycle sequenced usingThermoSequenase (Amersham Life Science). The KVLQT1 cosmids weresequenced by the dideoxy chain termination method on an AppliedBiosystems model 373A DNA sequencer. The exact exon/intron boundarieswere determined by comparison of cDNA, genomic sequences, and knownsplice site consensus sequences.

D. Design of PCR Primers and PCR Reaction Conditions

Primers to amplify exons of the two genes were designed empirically orusing OLIGO 4.0 (NBI). Amplification conditions were:

(1) 94° C. for 3 minutes followed by 30 cycles of 94° C. for 10 seconds,58° C. for 20 seconds and 72° C. for 20 seconds and a 5 minute extensionat 72° C.

(2) same as conditions in (1) except that the reactions had finalconcentrations of 10% glycerol and 4% formamide and were overlaid withmineral oil.

(3) 94° C. for 3 minutes followed by 5 cycles of 94° C. for 10 seconds,64° C. for 20 seconds and 72° C. for 20 seconds and 30 cycles of 94° C.for 10 seconds, 62° C. for 20 seconds and 72° C. for 20 seconds and a 5minute extension at 72° C.

E. KVLQT1 Genomic Structure and Primer Sets

Full length cDNA clones were isolated from an adult heart cDNA library.A 5′-cDNA probe generated from one of these clones was used to isolatecos1, a genomic cosmid clone containing exon 1. P1 genomic clonesencompassing the rest of the KVLQT1 cDNA were previously isolated (Wanget al., 1996). These genomic clones span approximately 400 kb onchromosome 11p15.5 (FIG. 6). To determine the exon structure andexon/intron boundaries, cos1 and P1 clones 118A10, 112E3, 46F10 and 49E5were sequenced using primers designed to the cDNA. Comparison of thegenomic and cDNA sequences of KVLQT1 revealed the presence of 16 exons(FIGS. 5A-5B and Table 3). Exon size ranged from 47 bp (exon 14) to 1122bp (exon 16). All intronic sequences contained the invariant GT and AGat the donor and acceptor splice sites, respectively (Table 3). One pairof PCR primers was designed for each of intron sequences flanking exons2 through 16 and two pairs of primers with overlapping products weredesigned for exon 1 due to its large size (Table 4). These primers canbe used to screen all KVLQT1 exons.

EXAMPLE 11 Characterization of KVLQT1 Function

To define the function of KVLQT1. Chinese hamster ovary (CHO) cells weretransfected with the complete cDNA described above in Example 9. TheKVLQT1 cDNA was subcloned into pCEP4 (InVitrogen). CHO cells werecultured in Ham's F-12 medium and transiently transfected usingLipofectamine (Gibco BRL). Cells were transfected for 18 hours in 35 mmdishes containing 6 μL lipofectamine, 0.5 μg green fluorescent protein(pGreen Lantern-1, Gibco BRL), and 1.5 μg of KVLQT1 in pCEP4.Fluorescent cells were voltage-clamped using an Axopatch 200 patch clampamplifier (Axon Instruments) 48 to 78 hours after transfection. Thebathing solution contained, in mM: 142 NaCl, 2 KCl, 1.2 MgCl₂, 1.8CaCl₂, 11.1 glucose, 5.5 HEPES buffer (pH 7.4. 22-25° C.). The pipettesolution contained, in mM: 110 potassium glutamate, 20 KCl, 1.0 MgCl₂, 5EGTA, 5 K₂ATP, 10 HEPES (pH 7.3). Data acquisition and analyses weredone using pCLAMP6 (Axon Instruments). The voltage dependence of currentactivation was determined by fitting the relationship between tailcurrents (determined by extrapolation of deactivating phase of currentto the end of the test pulse) and test potential with a Boltzmannfunction. Tail currents were normalized relative to the largest valuefor each oocyte.

TABLE 3 Intron/Exon Boundaries in KVLQT1 EXON Exon (total No.intron/EXON^(a) bases) EXON/intron^(a) 1 5′UTR . . . ATGGCCGCGG (9)  386+ ACTTCGCCGTgtgagtatcg (10) 2 tgtcttgcagCTTCCTCATC (11)  91CTTCTGGATGgtacgtagca (12) 3 gtccctgcagGAGATCGTGC (13) 127TCCATCATCGgtgagtcatg (14) 4 cactccacagACCTCATCGT (15)  79GGGCCATCAGgtgcgtctgt (16) 5 tccttcgcagGGGCATCCGC (17)  97CCACCGCCAGgtgggtggcc (18) 6 tctggcctagGAGCTGATAA (19) 141GTGGGGGGTGgtaagtcgga (20) 7 ctccctgcagGTCACAGTCA (21) 111GCTCCCAGCGgtaggtgccc (22) 8 tccttcccagGGGATTCTTG (23)  96ACTCATTCAGgtgcggtgcc (24) 9 cccacctcagACCGCATGGA (25) 123GTCTGTGGTGgtgagtagcc (26) 10 ttttttttagGTAAAGAAAA (27) 142GACAGTTCTGgtgagaaccc (28) 11 ttctcctcagTAAGGAAGAG (29) 121ACATCTCACAgtgagtgcct (30) 12 tccactgcagGCTGCGGGAA (31)  76GAAATTCCAGgtaagccctg (32) 13 tgtcccgcagCAAGCGCGGA (33)  95TGCAGAGGAGgtgggcacgg (34) 14 ttctctccagGCTGGACCAG (35)  47TCCGTCTCAGgtgggtttct (36) 15 tcccccatagAAAAGAGCAA (37)  62AGAAGACAAGgtaggctcac (38) 16 gtccccgcagGTGACGCAGC (39)   237+ GGGGTCCTGA. . . 3′UTR (40) ^(a)SEQ ID NO is shown in parentheses following eachsequence.

TABLE 4 Primers Used to Amplify KVLQT1 Exons Exon No. Forward Primer^(a)Reverse Primer^(a) Size C^(b) 1 CTCGCCTTCGCTGCAGCTC (41)GCGCGGGTCTAGGCTCACC (42) 334 2 1 CGCCGCGCCCCCAGTTGC (43)CAGAGCTCCCCCACACCAG (44) 224 2 2 ATGGGCAGAGGCCGTGATGCTGAC (45)ATCCAGCCATGCCCTCAGATGC (46) 165 3 3 GTTCAAACAGGTTGCAGGGTCTGA (47)CTTCCTGGTCTGGAAACCTGG (48) 256 3 4 CTCTTCCCTGGGGCCCTGGC (49)TGCGGGGGAGCTTGTGGCACAG (50) 170 3 5 TCAGCCCCACACCATCTCCTTC (51)CTGGGCCCCTACCCTAACCC (52) 154 3 6 TCCTGGAGCCCGACACTGTGTGT (53)TGTCCTGCCCACTCCTCAGCCT (54) 238 2 7 TGGCTGACCACTGTCCCTCT (55)CCCCAGGACCCCAGCTGTCCAA (56) 195 3 8 GCTGGCAGTGGCCTGTGTGGA (57)AACAGTGACCAAAATGACAGTGAC (58) 191 3 9 TGGCTCAGCAGGTGACAGC (59)TGGTGGCAGGTGGGCTACT (60) 185 1 10 GCCTGGCAGACGATGTCCA (61)CAACTGCCTGAGGGGTTCT (62) 216 1 11 CTGTCCCCACACTTTCTCCT (63)TGAGCTCCAGTCCCCTCCAG (64) 195 1 12 TGGCCACTCACAATCTCCT (65)GCCTTGACACCCTCCACTA (66) 222 1 13 GGCACAGGGAGGAGAAGTG (67)CGGCACCGCTGATCATGCA (68) 216 1 14 CCAGGGCCAGGTGTGACTG (69)TGGGCCCAGAGTAACTGACA (70) 119 2 15 GGCCCTGATTTGGGTGTTTTA (71)GGACGCTAACCAGAACCAC (72) 135 2 16 CACCACTGACTCTCTCGTCT (73)CCATCCCCCAGCCCCATC (74) 297 2 ^(a)SEQ ID NO is shown in parenthesesfollowing each sequence. ^(b)Conditions of the PCR as described inExample 10D.

A voltage-dependent, outward K⁺ current was observed after membranedepolarization to potentials above −60 mV (FIG. 7A). This currentreached a steady state within 1 second at +40 mV. Activation of thecurrent was preceded by a brief delay, and repolarization to −70 mVelicited a tail current with an initial increase in amplitude (a hook)before deactivation. Similar tail current hooks were previously observedfor HERG K⁻ channels, and were attributed to recovery of channels frominactivation at a rate faster than deactivation (Sanguinetti et al.,1995; Smith et al., 1996; Spector et al., 1996). The activation curvefor KVLQT1 current was half-maximal (V_(½)) at −11.6±0.6 mV, and had aslope factor of 12.6±0.5 mV (n=6; FIG. 7B).

The biophysical properties of KVLQT1 were unlike other known cardiac K⁺currents. It was hypothesized that KVLQT1 might coassemble with anothersubunit to form a known cardiac channel. The slowly activating delayedrectifier K⁺ current, I_(Ks), modulates repolarization of cardiac actionpotentials. Despite intensive study, the molecular structure of theI_(Ks) channel is not understood. Physiological data suggest that onecomponent of the I_(Ks) channel is minK (Goldstein and Miller, 1991;Hausdorff et al., 1991; Takumi et al., 1991; Busch et al., 1992; Wangand Goldstein, 1995; Wang et al., 1996), a 130 amino acid protein with asingle putative transmembrane domain (Takumi et al., 1988). The size andstructure of this protein, however, have led to doubt that minK aloneforms functional channels (Attali et al., 1993; Lesage et al., 1993).

To test this hypothesis, CHO cells were cotransfected with KVLQT1 andhuman KCNE1 cDNAs. A KCNE1 cDNA was subcloned in pCEP4 (InVitrogen) andtransfection was performed as described above for KVLQT1 alone. For thecotransfection of KVLQT1 and KCNE1, 0.75 μg of each cDNA was used. Asreported previously (Lesage et al., 1993), transfection of CHO cellswith KCNE1 alone did not induce detectable current (n=10, FIG. 7C).Cotransfection of KCNE1 with KVLQT1 induced a slowly activatingdelayed-rectifier current that was much larger than the current in cellstransfected with KVLQT1 alone (FIGS. 7D and 7E). The slow activation ofcurrent in cotransfected CHO cells was preceded by a delay that lastedseveral hundred msec, indicating that no significant homomeric KVLQT1channel current was present. Current did not saturate during longdepolarizing pulses, and required a three-exponential function to bestdescribe the initial delay and two phases of current activation. Duringa 30 sec depolarizing pulse to +40 mV, current was activated with timeconstants of 0.68±0.18, 1.48±0.16, and 8.0±0.6 sec (n=4). The isochronal(7.5 sec) activation curve for current had a V_(½) of 7.5±0.9 mV, and aslope factor of 16.5±0.8 mV (n=7; FIG. 9B). By comparison, the V_(½) andslope of the activation curve for human cardiac I_(Ks) are 9.4 mV and11.8 mV (Li et al., 1996). Like KVLQT1 and hminK coexpressed in CHOcells, activation of cardiac I_(Ks) is extremely slow and was bestdescribed by a three-exponential function (Balser et al., 1990;Sanguinetti and Jurkiewicz. 1990). Quinidine (50 μM) blocked tailcurrents in cotransfected CHO cells by 30 ±8% (n=5), similar to itseffect (40-50% block) on I_(Ks) in isolated myocytes (Balser et al,1991). Thus, coexpression of KVLQT1 and hminK in CHO cells induced a K⁻current with biophysical properties nearly identical to cardiac I_(Ks).

To characterize the properties of hminK and KVLQT1 further, thesechannels were expressed separately and together in Xenopus oocytes.Xenopus laevis oocytes were isolated and injected with cRNA as describedby Sanguinetti et al. (1995). KVLQT1 cDNA was subcloned into pSP64(Promega). KCNE1 cDNA was a gift from R. Swanson. Roughly equimolarconcentrations of KVLQT1 cRNA (5.8 ng per oocyte) and KCNE1 (1 ng peroocyte) cRNA were used for the co-injection experiments. The bathingsolution contained, in mM: 98 NaCl, 2 KCl, 2 MgCl₂, 0.1 CaCl₂, and 5HEPES (pH 7.6, 22-25° C.). For reversal-potential experiments,osmolarity was maintained by equimolar substitution of external NaCl forKCl. Currents were recorded using standard two-microelectrode voltageclamp techniques 3 days after injection of oocytes with cRNA(Sanguinetti et al., 1995). Currents were filtered at 0.5 kHz anddigitized at 2 kHz. Data are presented as mean±s.e.m.

Oocytes injected with KVLQT1 complementary RNA expressed a rapidlyactivating outward K⁻ current with a voltage dependence of activationnearly identical to CHO cells transfected with KVLQT1 cDNA (FIGS. 8A and8B). The K⁻ selectivity of KVLQT1 channels was determined by measuringthe reversal potential (E_(rev)) of tail currents in differentconcentrations of extracellular K ([K⁻]_(e)). The slope of therelationship between E_(rev) and log[K⁻]_(e) was 49.9±0.4 mV (n=7; FIG.8C), significantly less than predicted by the Nernst equation (58 mV)for a perfectly selective K⁺ channel. Co-injection of oocytes withKVLQT1 and KCNE1 cRNA induced a current similar to I_(Ks) (FIG. 9C). Theslope of the relationship between E_(rev) and log [K^(+]) _(e) forco-injected oocytes was 49.9±4 mV (n=6), similar to KVLQT1 alone and toguinea pig cardiac I_(Ks) (49 mV) (Matsuura et al., 1987). Theisochronal (7.5 sec) activation curve for co-injected oocytes had aV_(½) of 6.2 mV and a slope of 12.3 mV (FIG. 9E), similar to cardiacI_(Ks).

EXAMPLE 12 Identification of a KVLQT1 Gene in Xenopus

By contrast with CHO cells, KCNE1 was able to undergo functionalexpression in Xenopus oocytes (FIG. 9B). The induced current (I_(sK))was smaller than the current induced in co-injected oocytes, but thekinetics and voltage dependence of activation were similar (FIGS. 9A-E). Two observations have led to the hypothesis that I_(sK) in Xenopusoocytes results from channels formed by coassembly of minK With anunidentified, constitutively expressed subunit. First, the magnitude ofI_(sK) saturates after injection of very small amounts of KCNE1 cRNA(FIG. 9D), suggesting that an endogenous component of limited quantityis required for functional expression (Wang and Goldstein, 1995; Cui etal., 1994). Second, heterologous expression of minK in mammalian cellsdoes not induce detectable current.(Lesage et al., 1993) (FIG. 7C),suggesting that minK is not sufficient to form functional channels. Itwas hypothesized that this unidentified subunit might be a homologue ofKVLQT1. To test this hypothesis, a Xenopus oocyte cDNA library(Clontech) was screened with a KVLQT1 cDNA clone spanning the S3-S5domains. A 1.6 kb partial clone (XKVLQT1, FIG. 10A) was isolated.XKVLQT1 is 88% identical at the amino acid level with the correspondingregion of KVLQT1 (FIG. 10A). These data suggest that I_(sK) results fromthe coassembly of the XKVLQT1 and minK proteins.

It was concluded that KVLQT1 and hminK coassemble to form the cardiacI_(Ks) channel. Two delayed-rectifier K⁺ currents, I_(Kr) and I_(Ks),modulate action-potential duration in cardiac myocytes (Li et al., 1996;Sanguinetti and Jurkiewicz, 1990). Previous studies have implicateddysfunction of I_(Kr) channels in long QT syndrome (Sanguinetti et al.,1995; Curran et al., 1995; Sanguinetti et al., 1996a). The observationthat KVLQT1 mutations also cause this disorder (Wang et al., 1996), andthe discovery that KVLQT1 forms part of the I_(Ks) channel, indicatethat dysfunction of both cardiac delayed-rectifier K⁻ channelscontribute to risk of sudden death from cardiac arrhythmia.

EXAMPLE 13 Cosegregation of KVLQT1 Missense Mutations with LQT in SixLarge Families

To test the hypothesis that KVLQT1 is LQT1, single-strand conformationalpolymorphism (SSCP) analyses were used to screen for functionalmutations in affected members of K1532, the largest LQT family thatshowed linkage to chromosome 11. SSCP was carried out as previouslydescribed (Wang et al., 1995a; Wang et al., 1995b). Normal and aberrantSSCP products were isolated and sequenced directly as described (Wangand Keating, 1994) or subcloned into pBluescript (SK⁺) (Stratagene)using the T-vector method (Marchuk et al., 1991). When the latter methodwas used, several clones were sequenced by the dideoxy chain terminationmethod using Sequenase™ Version 2.0 (United States Biochemicals, Inc.).Analyses were focused on the region between S2 and S6 since theseregions might be important for KVLQT1 function. We designedoligonucleotide primers based on cDNA sequences and used these primersfor cycle sequencing reactions with the KVLQT1-containing P1, 18B12(Wang and Keating, 1994). These experiments defined intronic sequencesflanking exons encoding S2-S6. Additional primers were then generatedfrom these intronic sequences and used for SSCP analyses (Table 5).

SSCP analyses identified an anomalous conformer in the 70 affectedmembers of K1532 (FIG. 11A). This aberrant conformer was not observed inthe 147 unaffected members of this kindred or in genomic DNA from morethan 200 unrelated control individuals. The two-point LOD score forlinkage between this anomaly and LQT was 14.19 at a recombinationfraction of 0 (Table 2). No recombination was observed between KVLQT1and LQT1, indicating that these loci are completely linked. DNA sequenceanalyses of the normal and aberrant SSCP conformers revealed a singlebase substitution, a G to A transition, at the first nucleotide of codonVal-125 (FIG. 11A and Table 6). This mutation results in a valine tomethionine substitution in the predicted intracellular domain between S4and S5.

To further test the hypothesis that mutations in KVLQT1 cause LQT, DNAsamples from affected members of five additional large LQT kindreds werestudied. Linkage analyses with polymorphic markers from this region hadshown that the disease phenotype was linked to chromosome 11 in thesefamilies. Aberrant SSCP conformers were identified in affected membersof K2605, K1723, K1807 (FIGS. 11B-D), K161 and K162. The SSCP anomaliesidentified in K161 and K162 were identical to that observed in K1807.The aberrant SSCP conformer was not seen in unaffected members of thesekindreds or in DNA samples from more than 200 unrelated controlindividuals. The normal and aberrant conformers identified in eachfamily were sequenced. The nucleotide change, coding effect, andlocation of each mutation are summarized in Table 6.

TABLE 5 PCR Primers Used to Define KVLQT1 Mutations Region SEQ PrimerSequence Amplified ID NO: 1 GAGATCGTGCTGGTGGTGTTCT S2-S3 75 2CTTCCTGGTCTGGAAACCTGG 76 3 CTCTTCCCTGGGGCCCTGGC S3-S4 77 4TGCGGGGGAGCTTGTGGCACAG 78 5 GGGCATCCGCTTCCTGCAGA S4 79 6CTGGGCCCCTACCCTAACCC 80 7 TCCTGGAGCCCGAACTGTGTGT S5-Pore 81 8TGTCCTGCCCACTCCTCAGCCT 82 9 CCCCAGGACCCCAGCTGTCCAA Pore-S6 83 10AGGCTGACCACTGTCCCTCT 84 11 GCTGGCAGTGGCCTGTGTGGA S6 85 12AACAGTGACCAAAATGACAGTGAC 86

TABLE 6 Summary of KVLQT1 Mutations Nucleotide Coding No. of Codonchange effect Mutation Region Kindred affected 167-168 ΔTCG DeletionF167W/ S2 K13216 1 G168Δ 178 GCC to CCC Missense A178P S2-S3 K13119 1189 GGG to AGG Missense G189R S2-S3 K2557 3 190 CGG to CAG MissenseR190Q S2-S3 K15019 2 254 GTG to ATG Missense V254M S4-S5 K1532 70 273CTC to TTC Missense L273F S5 K1777 2 306 GGG to AGG Missense G306R PoreK20926 1 312 ACC to ATC Missense T312I Pore K20925 1 341 GCG to GAGMissense A341E S6 K1723 6 341 GCG to GAG Missense A341E S6 K2050 2 341GCG to GTG Missense A341V S6 K1807 6 341 GCG to GTG Missense A341V S6K161 18 341 GCG to GTG Missense A341V S6 K162 18 341 GCG to GTG MissenseA341V S6 K163 3 341 GCG to GTG Missense A341V S6 K164 2 345 GGG to GAGMissense G345E S6 K2605 11 168 GGG to AGG Missense G168R S2 K2625 — 168GGG to AGG Missense G168R S2 K2673 — 168 GGG to AGG Missense G168R S2K3698 — 314 GGC to AGC Missense G314S Pore K19187 — 315 TAT to TGTMissense Y315C Pore K22709 — 318 AAG to AAC Missense K318N Pore K2762 —353 CTG to CCG Missense L353P S6 K3401 — 366 CGG to TGG Missense R366WC-terminus K2824 —

EXAMPLE 14 A KVLQT1 Intragenic Deletion and Fifteen Missense MutationsAssociated with LQT in Small Families and Sporadic Cases

To identify additional LQT-associated mutations in KVLQT1, further SSCPanalyses were performed for small kindreds and sporadic cases. SSCPrevealed an aberrant conformer in kindred 13216 (FIG. 12A). Analyses ofmore than 200 unrelated control individuals failed to show this anomaly.This aberrant conformer was cloned and sequenced, revealing a three basepair deletion encompassing codons 38 and 39. This mutation results in aphenylalanine to tryptophan substitution and deletion of a glycine inthe putative S2 domain (Table 6).

Aberrant SSCP conformers were identified in affected members ofadditional kindreds. An aberrant SSCP conformer identified in K2050 wasidentical to that in K1723, and aberrant conformers identified in K161,K162, K163 and K164 were identical to that observed in K1807. Alsokindreds 2625, 2673 and 3698 had the identical mutation. None of theaberrant conformers was identified in DNA samples from more than 200control individuals. In each case, the normal and aberrant conformerswere sequenced. These data are shown in FIGS. 12A-O and summarized inTable 6. In total, KVLQT1 mutations associated with LQT in 24 familiesor sporadic cases were identified, providing strong molecular geneticevidence that mutations in KVLQT1 cause the chromosome 11-linked form ofLQT.

EXAMPLE 15 KCNE1 Variations Which Result in LQT

Separate studies on different individuals were performed in findingvariants of minK. These studies were performed using the followingmethods.

A. Phenotypic Analyses

Individuals were phenotypically characterized based on the QT intervalcorrected for heart rate. Individuals were characterized as affected ifQTc≧0.46 second. Individuals were assigned as unaffected if QTc≦0.42second. Informed consent was obtained from all individuals or theirguardians in accordance with local institutional review boardguidelines. Phenotypic data were interpreted without knowledge ofgenotype.

B. Mutation Analyses

Genomic samples were amplified by PCR using the following primer pairs:

MINK1F-5′-CTGCAGCAGTGGAACCTTAATG-3′(SEQ ID NO:87) and

MINK1R-5′-GTTCGAGTGCTCCAGCTTCTTG-3′(SEQ ID NO:88);

MINK2F-5′-AGGGCATCATGCTGAGCTACAT-3′(SEQ ID NO:89) and

MINK2R-5′-TTTAGCCAGTGGTGGGGTTCA-3′(SEQ ID NO:90);

MINK3F-5′-GTTCAGCAGGGTGGCAACAT-3′(SEQ ID NO:91) and

MINK3R-5′-GCCAGATGGTTTTCAACGACA-3′(SEQ ID NO:92).

PCR products were used in SSCP analysis as described (K W Wang et al.1996). PCR was completed with 75 ng DNA in a volume of 10 μL using aPerkin-Elmer Cetus 9600 thermocycler. Amplification conditions were 94°C. for 3 minutes followed by 30 cycles of 94° C. for 10 seconds, 58° C.for 20 seconds. 72° C. for 20 seconds and a 5 minute extension at 72° C.Reactions were diluted with 40 μL of 0.1% SDS/10 mM EDTA and with 30 μLof 95% formamide load dye. The mixture was denatured at 94° C. for 5minutes and placed on ice. Three microliters of each sample wereseparated on 5% and 10% non-denaturing polyacrylamide gels(acrylamide:bisacrylamide 49:1) at 4° C. and on 0.5× and 1×MDE (mutationdetection enhancement) gels (FMC BioProducts) at room temperature.Electrophoreses on the 5% and 10% gels were completed at 40 W for 3-5hours; electrophoreses on 0.5× and 1×MDE gels were completed overnight,respectively, at 350 V and 600 V. Gels were dried on 3 MM filter paperand exposed to film for 18 hours at −70° C.

SSCP bands were cut out of the gel and eluted in 100 μL double distilledwater at 65° C. for 30 minutes. Ten microliters of eluted DNA wasreamplified using the original primer pair. Products were separated on1% low melting temperature agarose gels (FMC), phenol-chloroformextracted and ethanol precipitated. DNA was sequenced in both directionsby the dideoxy chain termination method on an Applied Biosystems model373A DNA sequencer.

C. Functional Expression

KCNE1 cDNA expression constricts were amplified by PCR from total humanDNA and cloned in pSP64 transcription vector (Promega) using thefollowing primers:

MINKF-5′-CAGTGGAAGCTTAATGCCCAGGATGATC-3′(SEQ ID NO:93) and

MINKR-5′-CAGGAGGATCCAGTTTAGCCAGTGGTGGGGGTTCA-3′(SEQ ID NO:94).

Nucleotides in bold denote the changes made to create Hind III and BamHI restriction sites (underlined). A full-length KVLQT1 cDNA clone(identical to that reported by Yang et al. (1997)) was isolated from ahuman cardiac cDNA library and subcloned into the pSP64 plasmidexpression vector. All constructs were confirmed by DNA sequenceanalyses. Complementary RNAs were synthesized using the mCAP RNA cappingkit (Stratagene).

Isolation of Xenopus laevis oocytes and cRNA injection were performed asdescribed (Sanguinetti et al., 1995). Voltage clamp data were acquiredand analyzed using PCLAMP v6.0 software (Axon Instruments). Isochronal(7.5 seconds) rather than steady state measurements were used toestimate the voltage dependence of I_(Ks) activation. Thevoltage-dependence of I_(Ks) activation was determined by fitting peaktail currents to a Boltzmann function. V_(½), the voltage at which thecurrent was half-activated using this pulse protocol, and the slopefactor, were calculated from these data. Activating current was fittedto a biexponential function to obtain slow and fast time constants ofactivation. Deactivation time constants were obtained by fittingdecaying tail currents at various test potentials to a singleexponential function.

All data are mean±S.E.M. Statistical analyses were performed usingrepeated measures analysis of variance, with the Fisher's LeastSignificance post hoc test and the unpaired Student's T-test. A p value<0.05 was considered statistically significant.

D. Results

Ion channel β subunits are ancillary proteins that coassemble with αsubunits to modulate the gating kinetics and enhance stability ofmultimeric channel complexes (Rettig et al., 1994; Shi et al., 1996).Despite their functional importance, dysfunction of potassium β subunitshas not been associated with disease. Recent physiologic studies suggestthat KCNE1 encodes β subunits that coassemble with KvLQT1 α subunits toform the slowly activating delayed rectifier K⁺(I_(Ks)) channel(Sanguinetti et al., 1996b; Barhanin et al., 1996). Because KVLQT1mutations cause arrhythmia susceptibility in the long QT syndrome (LQT)(Q. Wang et al., 1996; Neyroud et al., 1997; Splawski et al., 1997a), wehypothesized that mutations in KCNE1 also cause this disorder. HereKCNE1 missense mutations are defined in affected members of two LQTfamilies. Both mutations (S74L, D76N) reduced I_(Ks) by shifting thevoltage dependence of activation and accelerating channel deactivation.D76N hminK also had a dominant negative effect. The functionalconsequences of these mutations would be delayed cardiac repolarizationand an increased risk of arrhythmia. These data establish KCNE1 as anLQT gene and confirm that hminK is an integral protein of the I_(Ks)channel.

Individuals with LQT have been ascertained and phenotypicallycharacterized (Keating et al., 1991a; Jiang et al., 1994). Single strandconformation polymorphism (SSCP) analyses using primers that span KCNE1led to the identification of an anomalous conformer in affected membersof kindred 1789 (FIG. 13A). This conformer was not observed inunaffected family members or in 200 unrelated control individuals (400chromosomes). DNA sequence analysis revealed a G to A transition at thefirst nucleotide of codon 76, causing an Asp to Asn substitution (D76N)(FIG. 13C). The sequences for KCNE1 cDNA and its protein product arelisted here as SEQ ID NO:3 and SEQ ID NO:4, respectively. The firstnucleotide of codon 76 is base 418 of SEQ ID NO:3.

Further SSCP analyses defined a second anomaly that cosegregated withthe disease in kindred 1754 (FIG. 13B). This anomaly was not observed inunaffected members of the family or in 200 controls. DNA sequenceanalysis revealed a C to T transition in the second nucleotide of codon74 (base 413 of SEQ ID NO:3), leading to substitution of Ser to Leu(S74L) (FIG. 13C). Analyses of further DNA samples obtained fromunrelated individuals with LQT revealed additional KCNE1 mutations.Table 7 lists the KCNE1 mutations found in LQT families.

TABLE 7 Summary of KCNE1 Mutations Codon Nucleotide change Coding effectMutation Kindred 28 TCG to TTG Missense S28L 1789 32 CGC to CAC MissenseR32H 2521 74 TCG to TTG Missense S74L 1754 76 GAC to AAC Missense D76N1789 98 CGG to TGG Missense R98W 2016 127 CCT to GCT Missense P127A 2016127 CCT to ACT Missense P127T 2819

To determine the functional consequences of these KCNE1 mutations, weexpressed mutant and wild-type (WT) proteins in Xenopus oocytes. Becausethe stoichiometry of KVLQT1 and minK interaction is not known, varyingamounts of KCNE1 cRNA (0.01-2.5 ng/oocyte) were coinjected with a fixedquantity of KPLQT1 cRNA (6 ng/oocyte) and the resultant currentsrecorded. I_(Ks) amplitude increased as a function of injected KCNE1,and saturated at KCNE1 cRNA levels ≧0.6 ng/oocyte (FIGS. 14A-14B).Subsequent coexpression experiments were performed using 1.2 ng/oocyteKCNE1 and 6 ng/oocyte KVLQT1 cRNA, to insure that KCNE1 was not alimiting factor for expression of heteromultimeric channels.

Coinjection of D76N KCNE1 and KPLQT1 cRNA failed to induce detectable K⁺currents (n=13). Because LQT is inherited as an autosomal dominanttrait, affected individuals possess one normal and one mutant KCNE1allele. Therefore, mutant KCNE1 cRNA was coinjected with WT KCNE1 andKVLQT1 cRNA. The current (I_(Ks-D76N)) induced by coinjection of D76NKCNE1 (0.6 ng/oocyte), WT KCNE1 (0.6 ng/oocyte) and KVLQT1 cRNA (6ng/oocyte) was 91% smaller than the current (I_(Ks-WT)) induced by WTKCNE1 (1.2 ng/oocyte) and KVLQT1 (6 ng/oocyte) cRNA at +40 mV (FIGS. 15Aand 15B). Thee data indicate that D76N hminK subunits formheteromultimeric channels with WT hminK and KVLQT1, and reduce I_(Ks) bya strong dominant-negative mechanism.

To compare the biophysical properties of wild-type and mutant channels,the voltage dependence of activation and the kinetics of deactivationfor I_(Ks-D76N) and I_(Ks-WT) were characterized. The magnitude ofI_(Ks) does not reach steady state even when elicited with pulses of 100second duration (Swanson et al., 1993). Therefore, tail currentamplitude following 7.5 second test pulses was used as an empiricalmeasure of the voltage dependence of I_(Ks). I_(Ks-D76N) tail currentswere half-maximal at +28 mV, a +16 mV shift relative to I_(Ks-WT) (FIG.15C). A shift in channel gating was confirmed by the voltage dependenceof current deactivation. The rate of I_(Ks-D76N) channel closure(deactivation) was faster than I_(KS-WT) at voltages ≧−80 mV (FIG. 15D).The voltage dependence of the time constants of deactivation wereshifted by approximately +30 mV. Thus, D76N hminK reduces I_(Ks) bythree mechanisms: a dominant negative suppression of channel function,an increased rate of channel deactivation and a positive shift in thevoltage dependence of channel activation. These effects would reduceoutward current during the repolarization phase and lengthen theduration of a cardiac action potential.

Unlike D76N hminK, S74L hminK formed I_(Ks) channels when coexpressedwith KVLQT1, albeit with altered function. Current induced by injectionof S74L KCNE1 (1.2 ng/oocyte) and KVLQT1 (6.0 ng/oocyte) cRNA had athreshold for activation that was approximately 40 mV higher thanI_(Ks-WT). The resultant current was 66% smaller than I_(Ks-WT) after7.5 second pulses to +60 mV (n=15). When S74L KCNE1 (0.6 ng/oocyte) andWT KCNE1 (0.6 ng/oocyte) were coinjected with KVLQT1 (6.0 ng/oocyte)cRNA, the resultant current (I_(Ks-S74L)) was reduced by approximately33% at +60 mV compared to I_(Ks-WT) (FIGS. 16A-16B). As shown in FIG.16C, this reduction was due primarily to a positive shift in the voltagedependence of current activation. The voltage dependence of deactivationwas shifted approximately +40 mV (FIG. 16D). This shift caused a markedincrease in the rate of I_(Ks-S74L) deactivation. Thus, S74L hminKsubunits form heteromultimeric channels with WT hminK and KVLQT1, andreduce I_(Ks) by a shift in the voltage dependence of channel activationand an increased rate of channel deactivation. Because I_(Ks-S74L) didnot equal I_(Ks-WT) at +60 mV (as expected for a simple shift ingating), it is possible tat S74L mutant subunits also reduce the numberof functional I_(Ks) channels and/or single channel conductance.

The observation that LQT-associated mutations of KCNE1 alter gatingkinetics provides compelling evidence that hminK forms an integral partof the I_(Ks) channel, rather than simply serving as a chaperone.Earlier studies of minK, performed before the discovery of KVLQT1, alsosupport this conclusion (Takumi et al., 1991; Goldstein and Miller,1991; Wang and Goldstein, 1995; K W Wang et al., 1996). In one of thesestudies, a mutant rat minK subunit (D77N), analogous to D76N hminK,coassembled with WT minK and suppressed I_(Ks) function, adominant-lethal effect (Wang and Goldstein, 1995).

It is concluded that mutations in KCNE1, the gene that encodes βsubunits of I_(Ks) channels, cause arrhythmia susceptibility by reducingI_(Ks) and thereby delaying myocellular repolarization. Because regionalheterogeneity in I_(Ks) exists within the myocardium (Liu andAntzelevitch, 1995), mutations in KCNE1 would cause abnormal regionaldisparity in action potential duration, creating a substrate forarrhythmia. The discovery of LQT-associated mutations in KCNE1 willfacilitate presymptomatic diagnosis of this disorder and may haveimplications for therapy.

EXAMPLE 16 Genomic Structure of KCNE1

The genomic DNA of KCNE1 was examined and the exon/intron boundariesdetermined for all exons essentially as done for KVLQT1. An adult heartcDNA library was screened with a PCR product amplified from total humanDNA and containing the entire coding sequence to isolate two identical1.7 kb KCNE1 clones. Two overlapping cosmid clones encompassing theentire KCNE1 cDNA were also isolated using full length KCNE1 as a probe(FIG. 17). The cosmids were sequenced by a dideoxy chain terminationmethod on an Applied Biosystems model 373A DNA sequencer to define thegenomic structure of the KCNE1 gene. Three exons comprise KCNE1 cDNA(FIG. 18 and Table 8). The two introns were located in the 5′-UTR. Thedonor and acceptor splice sites for both introns were GT and AG,respectively. Three pairs of primers were designed for screening KCNE1(Table 9). The first and second pair overlap and cover the entire codingsequence. The third pair amplifies part of the coding region includingthe putative transmembrane domain and some of the flanking sequences.

TABLE 8 Intron/Exon Boundaries in KCNE1 EXON Exon SIZE No.Intron/EXON^(a) (bp) EXON/Intron^(a) 1 5′UTR . . . CCACACCCG 33TCAGACCCGGgtgagttagg (95) (96) 2 caatcaccagGAAAAATCCC 111GGATATTCAGgtaggacctg (97) (98) 3 ttcctttaagAGGT . . . ATG 437 TTCCCCATGA. . . 3′UTR (99) (100) ^(a)SEQ ID NO is shown in parentheses followingeach sequence

TABLE 9 Primers Used to Amplify KCNE1 Coding Sequence Exon Size No.Forward Primer^(a) Reverse Primer^(a) (bp) C^(b) 3CTGCAGCAGTGGAACCTTAATG (101) GTTCGAGTGCTCCAGCTTCTTG (102) 264 1 3GGGCATCATGCTGAGCTACAT (103) TTTAGCCAGTGGTGGGGTTCA (104) 231 1 3GTTCAGCAGGGTGGCAACAT (105) GCCAGATGGTTTTCAACGACA (106) 281 1 ^(a)SEQ IDNO is shown in parentheses following each sequence. ^(b)Conditions ofthe PCR as described in Example 10D.

EXAMPLE 17 Generation of Polyclonal Antibody Against KVLQT1 or KCNE1

Segments of KVLQT1 or KCNE1 coding sequence are expressed as fusionprotein in E. coli. The overexpressed protein is purified by gel elutionand used to immunize rabbits and mice using a procedure similar to theone described by Harlow and Lane (1988). This procedure has been shownto generate Abs against various other proteins (for example, see Kraemeret al., 1993).

Briefly, a stretch of KVLQT1 or KCNE1 coding sequence is cloned as afusion protein in plasmid PET5A (Novagen, Inc., Madison, Wis.). Afterinduction with IPTG, the overexpression of a fusion protein with theexpected molecular weight is verified by SDS/PAGE. Fusion protein ispurified from the gel by electroelution. Identification of the proteinas the KVLQT1 or KCNE1 fusion product is verified by protein sequencingat the N-terminus. Next, the purified protein is used as immunogen inrabbits. Rabbits are immunized with 100 μg of the protein in completeFreund's adjuvant and boosted twice in 3 week intervals, first with 100μg of immunogen in incomplete Freund's adjuvant followed by 100 μg ofimmunogen in PBS. Antibody containing serum is collected two weeksthereafter.

This procedure is repeated to generate antibodies against the mutantforms of the KVLQT1 or KCNE1 gene product. These antibodies, inconjunction with antibodies to wild type KVLQT1 or KCNE1, are used todetect the presence and the relative level of the mutant forms invarious tissues and biological fluids.

EXAMPLE 18 Generation of Monoclonal Antibodies Specific for KVLQT1 orKCNE1

Monoclonal antibodies are generated according to the following protocol.Mice are immunized with immunogen comprising intact KVLQT1, KCNE1,KVLQT1 peptides or KCNE1 peptides (wild type or mutant) conjugated tokeyhole limpet hemocyanin using glutaraldehyde or EDC as is well known.

The immnmogen is mixed with an adjuvant. Each mouse receives fourinjections of 10 to 100 μg of immunogen and after the fourth injectionblood samples are taken from the mice to determine if the serum containsantibody to the immunogen. Serum titer is determined by ELISA or RIA.Mice with sera indicating the presence of antibody to the immunogen areselected for hybridoma production.

Spleens are removed from immune mice and a single cell suspension isprepared (see Harlow and Lane, 1988). Cell fusions are performedessentially as described by Kohler and Milstein (1975). Briefly, P3.65.3myeloma cells (American Type Culture Collection, Rockville, Md.) arefused with immune spleen cells using polyethylene glycol as described byHarlow and Lane (1988). Cells are plated at a density of 2×10⁵cells/well in 96 well tissue culture plates. Individual wells areexamined for growth and the supernatants of wells with growth are testedfor the presence of KVLQT1 or KCNE1 specific antibodies by ELISA or RIAusing wild type or mutant KVLQT1 or KCNE1 target protein. Cells inpositive wells are expanded and subcloned to establish and confirmmonoclonality.

Clones with the desired specificities are expanded and grown as ascitesin mice or in a hollow fiber system to produce sufficient quantities ofantibody for characterization and assay development.

EXAMPLE 19 Sandwich Assay for KVLQT1 or KCNE1

Monoclonal antibody is attached to a solid surface such as a plate,tube, bead or particle. Preferably, the antibody is attached to the wellsurface of a 96-well ELISA plate. 100 μL sample (e.g., serum, urine,tissue cytosol) containing the KVLQT1 or KCNE1 peptide/protein(wild-type or mutants) is added to the solid phase antibody. The sampleis incubated for 2 hrs at room temperature. Next the sample fluid isdecanted, and the solid phase is washed with buffer to remove unboundmaterial. 100 μL of a second monoclonal antibody (to a differentdeterminant on the KVLQT1 or KCNE1 peptide/protein) is added to thesolid phase. This antibody is labeled with a detector molecule (e.g.,¹²⁵I, enzyme, fluorophore, or a chromophore) and the solid phase withthe second antibody is incubated for two hrs at room temperature. Thesecond antibody is decanted and the solid phase is washed with buffer toremove unbound material.

The amount of bound label, which is proportional to the amount of KVLQT1or KCNE1 peptide/protein present in the sample, is quantified. Separateassays are performed using monoclonal antibodies which are specific forthe wild-type KVLQT1 or KCNE1 as well as monoclonal antibodies specificfor each of the mutations identified in KVLQT1 or KCNE1.

EXAMPLE 20 Assay to Screen Drugs Affecting the KVLQT1 and KCNE1 K⁺Channel

With the knowledge that KVLQT1 and KCNE1 coassemble to form a cardiacI_(Ks) a potassium channel, it is now possible to devise an assay toscreen for drugs which will have an effect on this channel. The twogenes, KVLQT1 and KCNE1, are cotransfected into oocytes or mammaliancells and coexpressed as described above. The cotransfection isperformed using any combination of wild-type or specifically mutatedKVLQT1 and KCNE1. When one of the genes used for cotransfection containsa mutation which causes LQT a change in the induced current is seen ascompared to cotransfection with wild-type genes only. A drug candidateis added to the bathing solution of the transfected cells to test theeffects of the drug candidates upon the induced current. A drugcandidate which alters the induced current such that it is closer to thecurrent seen with cells cotransfected with wild-type KVLQT1 and KCNE1 isuseful for treating LQT.

While the invention has been disclosed in this patent application byreference to the details of preferred embodiments of the invention, itis to be understood that the disclosure is intended in an illustrativerather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

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114 1 3181 DNA Homo sapiens CDS (163)..(2190) 1 ctgccccctc cggccccgccccgagcgccc gggctgggcc ggcagcggcc ccccgcggcg 60 gggctggcag cagtggctgcccgcactgcg cccgggcgct cgccttcgct gcagctcccg 120 gtgccgccgc tcgggccggccccccggcag gccctcctcg tt atg gcc gcg gcc 174 Met Ala Ala Ala 1 tcc tccccg ccc agg gcc gag agg aag cgc tgg ggt tgg ggc cgc ctg 222 Ser Ser ProPro Arg Ala Glu Arg Lys Arg Trp Gly Trp Gly Arg Leu 5 10 15 20 cca ggcgcc cgg cgg ggc agc gcg ggc ctg gcc aag aag tgc ccc ttc 270 Pro Gly AlaArg Arg Gly Ser Ala Gly Leu Ala Lys Lys Cys Pro Phe 25 30 35 tcg ctg gagctg gcg gag ggc ggc ccg gcg ggc ggc gcg ctc tac gcg 318 Ser Leu Glu LeuAla Glu Gly Gly Pro Ala Gly Gly Ala Leu Tyr Ala 40 45 50 ccc atc gcg cccggc gcc cca ggt ccc gcg ccc cct gcg tcc ccg gcc 366 Pro Ile Ala Pro GlyAla Pro Gly Pro Ala Pro Pro Ala Ser Pro Ala 55 60 65 gcg ccc gcc gcg ccccca gtt gcc tcc gac ctt ggc ccg cgg ccg ccg 414 Ala Pro Ala Ala Pro ProVal Ala Ser Asp Leu Gly Pro Arg Pro Pro 70 75 80 gtg agc cta gac ccg cgcgtc tcc atc tac agc acg cgc cgc ccg gtg 462 Val Ser Leu Asp Pro Arg ValSer Ile Tyr Ser Thr Arg Arg Pro Val 85 90 95 100 ttg gcg cgc acc cac gtccag ggc cgc gtc tac aac ttc ctc gag cgt 510 Leu Ala Arg Thr His Val GlnGly Arg Val Tyr Asn Phe Leu Glu Arg 105 110 115 ccc acc ggc tgg aaa tgcttc gtt tac cac ttc gcc gtc ttc ctc atc 558 Pro Thr Gly Trp Lys Cys PheVal Tyr His Phe Ala Val Phe Leu Ile 120 125 130 gtc ctg gtc tgc ctc atcttc agc gtg ctg tcc acc atc gag cag tat 606 Val Leu Val Cys Leu Ile PheSer Val Leu Ser Thr Ile Glu Gln Tyr 135 140 145 gcc gcc ctg gcc acg gggact ctc ttc tgg atg gag atc gtg ctg gtg 654 Ala Ala Leu Ala Thr Gly ThrLeu Phe Trp Met Glu Ile Val Leu Val 150 155 160 gtg ttc ttc ggg acg gagtac gtg gtc cgc ctc tgg tcc gcc ggc tgc 702 Val Phe Phe Gly Thr Glu TyrVal Val Arg Leu Trp Ser Ala Gly Cys 165 170 175 180 cgc agc aag tac gtgggc ctc tgg ggg cgg ctg cgc ttt gcc cgg aag 750 Arg Ser Lys Tyr Val GlyLeu Trp Gly Arg Leu Arg Phe Ala Arg Lys 185 190 195 ccc att tcc atc atcgac ctc atc gtg gtc gtg gcc tcc atg gtg gtc 798 Pro Ile Ser Ile Ile AspLeu Ile Val Val Val Ala Ser Met Val Val 200 205 210 ctc tgc gtg ggc tccaag ggg cag gtg ttt gcc acg tcg gcc atc agg 846 Leu Cys Val Gly Ser LysGly Gln Val Phe Ala Thr Ser Ala Ile Arg 215 220 225 ggc atc cgc ttc ctgcag atc ctg agg atg cta cac gtc gac cgc cag 894 Gly Ile Arg Phe Leu GlnIle Leu Arg Met Leu His Val Asp Arg Gln 230 235 240 gga ggc acc tgg aggctc ctg ggc tcc gtg gtc ttc atc cac cgc cag 942 Gly Gly Thr Trp Arg LeuLeu Gly Ser Val Val Phe Ile His Arg Gln 245 250 255 260 gag ctg ata accacc ctg tac atc ggc ttc ctg ggc ctc atc ttc tcc 990 Glu Leu Ile Thr ThrLeu Tyr Ile Gly Phe Leu Gly Leu Ile Phe Ser 265 270 275 tcg tac ttt gtgtac ctg gct gag aag gac gcg gtg aac gag tca ggc 1038 Ser Tyr Phe Val TyrLeu Ala Glu Lys Asp Ala Val Asn Glu Ser Gly 280 285 290 cgc gtg gag ttcggc agc tac gca gat gcg ctg tgg tgg ggg gtg gtc 1086 Arg Val Glu Phe GlySer Tyr Ala Asp Ala Leu Trp Trp Gly Val Val 295 300 305 aca gtc acc accatc ggc tat ggg gac aag gtg ccc cag acg tgg gtc 1134 Thr Val Thr Thr IleGly Tyr Gly Asp Lys Val Pro Gln Thr Trp Val 310 315 320 ggg aag acc atcgcc tcc tgc ttc tct gtc ttt gcc atc tcc ttc ttt 1182 Gly Lys Thr Ile AlaSer Cys Phe Ser Val Phe Ala Ile Ser Phe Phe 325 330 335 340 gcg ctc ccagcg ggg att ctt ggc tcg ggg ttt gcc ctg aag gtg cag 1230 Ala Leu Pro AlaGly Ile Leu Gly Ser Gly Phe Ala Leu Lys Val Gln 345 350 355 cag aag cagagg cag aag cac ttc aac cgg cag atc ccg gcg gca gcc 1278 Gln Lys Gln ArgGln Lys His Phe Asn Arg Gln Ile Pro Ala Ala Ala 360 365 370 tca ctc attcag acc gca tgg agg tgc tat gct gcc gag aac ccc gac 1326 Ser Leu Ile GlnThr Ala Trp Arg Cys Tyr Ala Ala Glu Asn Pro Asp 375 380 385 tcc tcc acctgg aag atc tac atc cgg aag gcc ccc cgg agc cac act 1374 Ser Ser Thr TrpLys Ile Tyr Ile Arg Lys Ala Pro Arg Ser His Thr 390 395 400 ctg ctg tcaccc agc ccc aaa ccc aag aag tct gtg gtg gta aag aaa 1422 Leu Leu Ser ProSer Pro Lys Pro Lys Lys Ser Val Val Val Lys Lys 405 410 415 420 aaa aagttc aag ctg gac aaa gac aat ggg gtg act cct gga gag aag 1470 Lys Lys PheLys Leu Asp Lys Asp Asn Gly Val Thr Pro Gly Glu Lys 425 430 435 atg ctcaca gtc ccc cat atc acg tgc gac ccc cca gaa gag cgg cgg 1518 Met Leu ThrVal Pro His Ile Thr Cys Asp Pro Pro Glu Glu Arg Arg 440 445 450 ctg gaccac ttc tct gtc gac ggc tat gac agt tct gta agg aag agc 1566 Leu Asp HisPhe Ser Val Asp Gly Tyr Asp Ser Ser Val Arg Lys Ser 455 460 465 cca acactg ctg gaa gtg agc atg ccc cat ttc atg aga acc aac agc 1614 Pro Thr LeuLeu Glu Val Ser Met Pro His Phe Met Arg Thr Asn Ser 470 475 480 ttc gccgag gac ctg gac ctg gaa ggg gag act ctg ctg aca ccc atc 1662 Phe Ala GluAsp Leu Asp Leu Glu Gly Glu Thr Leu Leu Thr Pro Ile 485 490 495 500 acccac atc tca cag ctg cgg gaa cac cat cgg gcc acc att aag gtc 1710 Thr HisIle Ser Gln Leu Arg Glu His His Arg Ala Thr Ile Lys Val 505 510 515 attcga cgc atg cag tac ttt gtg gcc aag aag aaa ttc cag caa gcg 1758 Ile ArgArg Met Gln Tyr Phe Val Ala Lys Lys Lys Phe Gln Gln Ala 520 525 530 cggaag cct tac gat gtg cgg gac gtc att gag cag tac tcg cag ggc 1806 Arg LysPro Tyr Asp Val Arg Asp Val Ile Glu Gln Tyr Ser Gln Gly 535 540 545 cacctc aac ctc atg gtg cgc atc aag gag ctg cag agg agg ctg gac 1854 His LeuAsn Leu Met Val Arg Ile Lys Glu Leu Gln Arg Arg Leu Asp 550 555 560 cagtcc att ggg aag ccc tca ctg ttc atc tcc gtc tca gaa aag agc 1902 Gln SerIle Gly Lys Pro Ser Leu Phe Ile Ser Val Ser Glu Lys Ser 565 570 575 580aag gat cgc ggc agc aac acg atc ggc gcc cgc ctg aac cga gta gaa 1950 LysAsp Arg Gly Ser Asn Thr Ile Gly Ala Arg Leu Asn Arg Val Glu 585 590 595gac aag gtg acg cag ctg gac cag agg ctg gca ctc atc acc gac atg 1998 AspLys Val Thr Gln Leu Asp Gln Arg Leu Ala Leu Ile Thr Asp Met 600 605 610ctt cac cag ctg ctc tcc ttg cac ggt ggc agc acc ccc ggc agc ggc 2046 LeuHis Gln Leu Leu Ser Leu His Gly Gly Ser Thr Pro Gly Ser Gly 615 620 625ggc ccc ccc aga gag ggc ggg gcc cac atc acc cag ccc tgc ggc agt 2094 GlyPro Pro Arg Glu Gly Gly Ala His Ile Thr Gln Pro Cys Gly Ser 630 635 640ggc ggc tcc gtc gac cct gag ctc ttc ctg ccc agc aac acc ctg ccc 2142 GlyGly Ser Val Asp Pro Glu Leu Phe Leu Pro Ser Asn Thr Leu Pro 645 650 655660 acc tac gag cag ctg acc gtg ccc agg agg ggc ccc gat gag ggg tcc 2190Thr Tyr Glu Gln Leu Thr Val Pro Arg Arg Gly Pro Asp Glu Gly Ser 665 670675 tgaggagggg atggggctgg gggatgggcc tgagtgagag gggaggccaa gagtggcccc2250 acctggccct ctctgaagga ggccacctcc taaaaggccc agagagaaga gccccactct2310 cagaggcccc aataccccat ggaccatgct gtctggcaca gcctgcactt gggggctcag2370 caaggccacc tcttcctggc cggtgtgggg gccccgtctc aggtctgagt tgttacccca2430 agcgccctgg cccccacatg gtgatgttga catcactggc atggtggttg ggacccagtg2490 gcagggcaca gggcctggcc catgtatggc caggaagtag cacaggctga gtgcaggccc2550 accctgcttg gcccaggggg cttcctgagg ggagacagag caacccctgg accccagcct2610 caaatccagg accctgccag gcacaggcag ggcaggacca gcccacgctg actacagggc2670 caccggcaat aaaagcccag gagcccattt ggagggcctg ggcctggctc cctcactctc2730 aggaaatgct gacccatggg caggagactg tggagactgc tcctgagccc ccagcttcca2790 gcaggaggga cagtctcacc atttccccag ggcacgtggt tgagtggggg gaacgcccac2850 ttccctgggt tagactgcca gctcttccta gctggagagg agccctgcct ctccgcccct2910 gagcccactg tgcgtggggc tcccgcctcc aacccctcgc ccagtcccag cagccagcca2970 aacacacaga aggggactgc cacctcccct tgccagctgc tgagccgcag agaagtgacg3030 gttcctacac aggacagggg ttccttctgg gcattacatc gcatagaaat caataatttg3090 tggtgatttg gatctgtgtt ttaatgagtt tcacagtgtg attttgatta ttaattgtgc3150 aagcttttcc taataaacgt ggagaatcac a 3181 2 676 PRT Homo sapiens 2Met Ala Ala Ala Ser Ser Pro Pro Arg Ala Glu Arg Lys Arg Trp Gly 1 5 1015 Trp Gly Arg Leu Pro Gly Ala Arg Arg Gly Ser Ala Gly Leu Ala Lys 20 2530 Lys Cys Pro Phe Ser Leu Glu Leu Ala Glu Gly Gly Pro Ala Gly Gly 35 4045 Ala Leu Tyr Ala Pro Ile Ala Pro Gly Ala Pro Gly Pro Ala Pro Pro 50 5560 Ala Ser Pro Ala Ala Pro Ala Ala Pro Pro Val Ala Ser Asp Leu Gly 65 7075 80 Pro Arg Pro Pro Val Ser Leu Asp Pro Arg Val Ser Ile Tyr Ser Thr 8590 95 Arg Arg Pro Val Leu Ala Arg Thr His Val Gln Gly Arg Val Tyr Asn100 105 110 Phe Leu Glu Arg Pro Thr Gly Trp Lys Cys Phe Val Tyr His PheAla 115 120 125 Val Phe Leu Ile Val Leu Val Cys Leu Ile Phe Ser Val LeuSer Thr 130 135 140 Ile Glu Gln Tyr Ala Ala Leu Ala Thr Gly Thr Leu PheTrp Met Glu 145 150 155 160 Ile Val Leu Val Val Phe Phe Gly Thr Glu TyrVal Val Arg Leu Trp 165 170 175 Ser Ala Gly Cys Arg Ser Lys Tyr Val GlyLeu Trp Gly Arg Leu Arg 180 185 190 Phe Ala Arg Lys Pro Ile Ser Ile IleAsp Leu Ile Val Val Val Ala 195 200 205 Ser Met Val Val Leu Cys Val GlySer Lys Gly Gln Val Phe Ala Thr 210 215 220 Ser Ala Ile Arg Gly Ile ArgPhe Leu Gln Ile Leu Arg Met Leu His 225 230 235 240 Val Asp Arg Gln GlyGly Thr Trp Arg Leu Leu Gly Ser Val Val Phe 245 250 255 Ile His Arg GlnGlu Leu Ile Thr Thr Leu Tyr Ile Gly Phe Leu Gly 260 265 270 Leu Ile PheSer Ser Tyr Phe Val Tyr Leu Ala Glu Lys Asp Ala Val 275 280 285 Asn GluSer Gly Arg Val Glu Phe Gly Ser Tyr Ala Asp Ala Leu Trp 290 295 300 TrpGly Val Val Thr Val Thr Thr Ile Gly Tyr Gly Asp Lys Val Pro 305 310 315320 Gln Thr Trp Val Gly Lys Thr Ile Ala Ser Cys Phe Ser Val Phe Ala 325330 335 Ile Ser Phe Phe Ala Leu Pro Ala Gly Ile Leu Gly Ser Gly Phe Ala340 345 350 Leu Lys Val Gln Gln Lys Gln Arg Gln Lys His Phe Asn Arg GlnIle 355 360 365 Pro Ala Ala Ala Ser Leu Ile Gln Thr Ala Trp Arg Cys TyrAla Ala 370 375 380 Glu Asn Pro Asp Ser Ser Thr Trp Lys Ile Tyr Ile ArgLys Ala Pro 385 390 395 400 Arg Ser His Thr Leu Leu Ser Pro Ser Pro LysPro Lys Lys Ser Val 405 410 415 Val Val Lys Lys Lys Lys Phe Lys Leu AspLys Asp Asn Gly Val Thr 420 425 430 Pro Gly Glu Lys Met Leu Thr Val ProHis Ile Thr Cys Asp Pro Pro 435 440 445 Glu Glu Arg Arg Leu Asp His PheSer Val Asp Gly Tyr Asp Ser Ser 450 455 460 Val Arg Lys Ser Pro Thr LeuLeu Glu Val Ser Met Pro His Phe Met 465 470 475 480 Arg Thr Asn Ser PheAla Glu Asp Leu Asp Leu Glu Gly Glu Thr Leu 485 490 495 Leu Thr Pro IleThr His Ile Ser Gln Leu Arg Glu His His Arg Ala 500 505 510 Thr Ile LysVal Ile Arg Arg Met Gln Tyr Phe Val Ala Lys Lys Lys 515 520 525 Phe GlnGln Ala Arg Lys Pro Tyr Asp Val Arg Asp Val Ile Glu Gln 530 535 540 TyrSer Gln Gly His Leu Asn Leu Met Val Arg Ile Lys Glu Leu Gln 545 550 555560 Arg Arg Leu Asp Gln Ser Ile Gly Lys Pro Ser Leu Phe Ile Ser Val 565570 575 Ser Glu Lys Ser Lys Asp Arg Gly Ser Asn Thr Ile Gly Ala Arg Leu580 585 590 Asn Arg Val Glu Asp Lys Val Thr Gln Leu Asp Gln Arg Leu AlaLeu 595 600 605 Ile Thr Asp Met Leu His Gln Leu Leu Ser Leu His Gly GlySer Thr 610 615 620 Pro Gly Ser Gly Gly Pro Pro Arg Glu Gly Gly Ala HisIle Thr Gln 625 630 635 640 Pro Cys Gly Ser Gly Gly Ser Val Asp Pro GluLeu Phe Leu Pro Ser 645 650 655 Asn Thr Leu Pro Thr Tyr Glu Gln Leu ThrVal Pro Arg Arg Gly Pro 660 665 670 Asp Glu Gly Ser 675 3 1703 DNA Homosapiens CDS (193)..(579) 3 acacccggct ctctcggcat ctcagacccg ggaaaaatccctctgctttc tctggccagt 60 ttcacacaat catcaggtga gccgaggatc cattggaggaaggcattatc tgtatccaga 120 ggaaatagcc aaggatattc agaggtgtgc ctgggaagtttgagctgcag cagtggaacc 180 ttaatgccca gg atg atc ctg tct aac acc aca gcggtg acg ccc ttt ctg 231 Met Ile Leu Ser Asn Thr Thr Ala Val Thr Pro PheLeu 1 5 10 acc aag ctg tgg cag gag aca gtt cag cag ggt ggc aac atg tcgggc 279 Thr Lys Leu Trp Gln Glu Thr Val Gln Gln Gly Gly Asn Met Ser Gly15 20 25 ctg gcc cgc agg tcc ccc cgc agc ggt gac ggc aag ctg gag gcc ctc327 Leu Ala Arg Arg Ser Pro Arg Ser Gly Asp Gly Lys Leu Glu Ala Leu 3035 40 45 tac gtc ctc atg gta ctg gga ttc ttc ggc ttc ttc acc ctg ggc atc375 Tyr Val Leu Met Val Leu Gly Phe Phe Gly Phe Phe Thr Leu Gly Ile 5055 60 atg ctg agc tac atc cgc tcc aag aag ctg gag cac tcg aac gac cca423 Met Leu Ser Tyr Ile Arg Ser Lys Lys Leu Glu His Ser Asn Asp Pro 6570 75 ttc aac gtc tac atc gag tcc gat gcc tgg caa gag aag gac aag gcc471 Phe Asn Val Tyr Ile Glu Ser Asp Ala Trp Gln Glu Lys Asp Lys Ala 8085 90 tat gtc cag gcc cgg gtc ctg gag agc tac agg tcg tgc tat gtc gtt519 Tyr Val Gln Ala Arg Val Leu Glu Ser Tyr Arg Ser Cys Tyr Val Val 95100 105 gaa aac cat ctg gcc ata gaa caa ccc aac aca cac ctt cct gag acg567 Glu Asn His Leu Ala Ile Glu Gln Pro Asn Thr His Leu Pro Glu Thr 110115 120 125 aag cct tcc cca tgaaccccac cactggctaa actggacacc tcctgctggn619 Lys Pro Ser Pro nnnnagattt tctaatcaca ttcctctcat actctttattgtgatggata ccactggatt 679 tctttttggc tgttgtaang ggtgaggggt ggattaatgacactgtttca ctgtttctct 739 aaaatcacgt tcttttgtga tagactgtca gtggttcccccatatctgtc cctgccttgc 799 taaatttagc agaatccctg aggacatggc ctctgagaatagcagctgca tttcccagac 859 tcccttgcag ctagcaaggt tgtgtgacta agccctggccagtaggcatg gaagtgaaga 919 ctgtaatgtc caagtaatcc ttggaaagaa aagaacgtgcccttaactaa ctttgtcctg 979 cttcccagtg gctggatgtg gaggaggtgg agagcagttatgagactggg aaagttcggg 1039 gcactcaaag agccacacac atctgggcct gggcgacgtggatcctcctt accacccacc 1099 aggccagatt tacaggagag agaaatccac tccactcttccttaagccac tgttattctg 1159 atctctgtta aggtcgcaga atcaatgccc ttactgatacacctacctta taggactgaa 1219 cctaaaggca tgacatttcc atacttgtca caagcacacactgattctgc ccttgtcact 1279 tctgtgctca ctcttgtggc tctatcctcc tcctgcccttccgccttcca ctcctccctt 1339 gcacccatcc tgcacacatc tccctgaaaa cacacaggcacatacactca tatacataga 1399 cacacataca cacctcaatc tagaaagaac ttgctttgtacagggctgag atggaggaga 1459 aaaaaatgcc cccttcagaa tgcataccaa ggggaaggtgctcggtcact gtgggagcag 1519 ggaaaggtgc ccccactccc cgagagccag gggaaggagtggctctgggc agagagggac 1579 acatagcact ggggtggcag gtccttttga ggtgatgggccggttttgtg agatgaattg 1639 tatcccccaa aaagacaggt accttcaatg tgacctaattgggaaataga gtctttgcag 1699 atga 1703 4 129 PRT Homo sapiens 4 Met IleLeu Ser Asn Thr Thr Ala Val Thr Pro Phe Leu Thr Lys Leu 1 5 10 15 TrpGln Glu Thr Val Gln Gln Gly Gly Asn Met Ser Gly Leu Ala Arg 20 25 30 ArgSer Pro Arg Ser Gly Asp Gly Lys Leu Glu Ala Leu Tyr Val Leu 35 40 45 MetVal Leu Gly Phe Phe Gly Phe Phe Thr Leu Gly Ile Met Leu Ser 50 55 60 TyrIle Arg Ser Lys Lys Leu Glu His Ser Asn Asp Pro Phe Asn Val 65 70 75 80Tyr Ile Glu Ser Asp Ala Trp Gln Glu Lys Asp Lys Ala Tyr Val Gln 85 90 95Ala Arg Val Leu Glu Ser Tyr Arg Ser Cys Tyr Val Val Glu Asn His 100 105110 Leu Ala Ile Glu Gln Pro Asn Thr His Leu Pro Glu Thr Lys Pro Ser 115120 125 Pro 5 63 DNA Artificial Sequence Description of ArtificialSequenceHypothetical sequence to demonstrate calculation of percenthomology or identity. 5 accgtagcta cgtacgtata tagaaagggc gcgatcgtcgtcgcgtatga cgacttagca 60 tgc 63 6 130 DNA Artificial SequenceDescription of Artificial SequenceHypothetical sequence to demonstratecalculation of percent homology or identity. 6 accggtagct acgtacgttatttagaaagg ggtgtgtgtg tgtgtgtaaa ccggggtttt 60 cgggatcgtc cgtcgcgtatgacgacttag ccatgcacgg tatatcgtat taggactagc 120 gattgactag 130 7 17 DNAHomo sapiens 7 cagatcctga ggatgct 17 8 17 DNA Homo sapiens 8 gtacctggctgagaagg 17 9 10 DNA Homo sapiens 9 atggccgcgg 10 10 20 DNA Homo sapiens10 acttcgccgt gtgagtatcg 20 11 20 DNA Homo sapiens 11 tgtcttgcagcttcctcatc 20 12 20 DNA Homo sapiens 12 cttctggatg gtacgtagca 20 13 20DNA Homo sapiens 13 gtccctgcag gagatcgtgc 20 14 20 DNA Homo sapiens 14tccatcatcg gtgagtcatg 20 15 20 DNA Homo sapiens 15 cactccacag acctcatcgt20 16 20 DNA Homo sapiens 16 gggccatcag gtgcgtctgt 20 17 20 DNA Homosapiens 17 tccttcgcag gggcatccgc 20 18 20 DNA Homo sapiens 18 ccaccgccaggtgggtggcc 20 19 20 DNA Homo sapiens 19 tctggcctag gagctgataa 20 20 20DNA Homo sapiens 20 gtggggggtg gtaagtcgga 20 21 20 DNA Homo sapiens 21ctccctgcag gtcacagtca 20 22 20 DNA Homo sapiens 22 gctcccagcg gtaggtgccc20 23 20 DNA Homo sapiens 23 tccttcccag gggattcttg 20 24 20 DNA Homosapiens 24 actcattcag gtgcggtgcc 20 25 20 DNA Homo sapiens 25 cccacctcagaccgcatgga 20 26 20 DNA Homo sapiens 26 gtctgtggtg gtgagtagcc 20 27 20DNA Homo sapiens 27 ttttttttag gtaaagaaaa 20 28 20 DNA Homo sapiens 28gacagttctg gtgagaaccc 20 29 20 DNA Homo sapiens 29 ttctcctcag taaggaagag20 30 20 DNA Homo sapiens 30 acatctcaca gtgagtgcct 20 31 20 DNA Homosapiens 31 tccactgcag gctgcgggaa 20 32 20 DNA Homo sapiens 32 gaaattccaggtaagccctg 20 33 20 DNA Homo sapiens 33 tgtcccgcag caagcgcgga 20 34 20DNA Homo sapiens 34 tgcagaggag gtgggcacgg 20 35 20 DNA Homo sapiens 35ttctctccag gctggaccag 20 36 20 DNA Homo sapiens 36 tccgtctcag gtgggtttct20 37 20 DNA Homo sapiens 37 tcccccatag aaaagagcaa 20 38 20 DNA Homosapiens 38 agaagacaag gtaggctcac 20 39 20 DNA Homo sapiens 39 gtccccgcaggtgacgcagc 20 40 10 DNA Homo sapiens 40 ggggtcctga 10 41 19 DNA Homosapiens 41 ctcgccttcg ctgcagctc 19 42 19 DNA Homo sapiens 42 gcgcgggtctaggctcacc 19 43 18 DNA Homo sapiens 43 cgccgcgccc ccagttgc 18 44 19 DNAHomo sapiens 44 cagagctccc ccacaccag 19 45 24 DNA Homo sapiens 45atgggcagag gccgtgatgc tgac 24 46 22 DNA Homo sapiens 46 atccagccatgccctcagat gc 22 47 24 DNA Homo sapiens 47 gttcaaacag gttgcagggt ctga 2448 21 DNA Homo sapiens 48 cttcctggtc tggaaacctg g 21 49 20 DNA Homosapiens 49 ctcttccctg gggccctggc 20 50 22 DNA Homo sapiens 50 tgcgggggagcttgtggcac ag 22 51 22 DNA Homo sapiens 51 tcagccccac accatctcct tc 2252 20 DNA Homo sapiens 52 ctgggcccct accctaaccc 20 53 23 DNA Homosapiens 53 tcctggagcc cgacactgtg tgt 23 54 22 DNA Homo sapiens 54tgtcctgccc actcctcagc ct 22 55 20 DNA Homo sapiens 55 tggctgaccactgtccctct 20 56 22 DNA Homo sapiens 56 ccccaggacc ccagctgtcc aa 22 5721 DNA Homo sapiens 57 gctggcagtg gcctgtgtgg a 21 58 24 DNA Homo sapiens58 aacagtgacc aaaatgacag tgac 24 59 19 DNA Homo sapiens 59 tggctcagcaggtgacagc 19 60 19 DNA Homo sapiens 60 tggtggcagg tgggctact 19 61 19 DNAHomo sapiens 61 gcctggcaga cgatgtcca 19 62 19 DNA Homo sapiens 62caactgcctg aggggttct 19 63 20 DNA Homo sapiens 63 ctgtccccac actttctcct20 64 20 DNA Homo sapiens 64 tgagctccag tcccctccag 20 65 19 DNA Homosapiens 65 tggccactca caatctcct 19 66 19 DNA Homo sapiens 66 gccttgacaccctccacta 19 67 19 DNA Homo sapiens 67 ggcacaggga ggagaagtg 19 68 19 DNAHomo sapiens 68 cggcaccgct gatcatgca 19 69 19 DNA Homo sapiens 69ccagggccag gtgtgactg 19 70 20 DNA Homo sapiens 70 tgggcccaga gtaactgaca20 71 21 DNA Homo sapiens 71 ggccctgatt tgggtgtttt a 21 72 19 DNA Homosapiens 72 ggacgctaac cagaaccac 19 73 20 DNA Homo sapiens 73 caccactgactctctcgtct 20 74 18 DNA Homo sapiens 74 ccatccccca gccccatc 18 75 22 DNAHomo sapiens 75 gagatcgtgc tggtggtgtt ct 22 76 21 DNA Homo sapiens 76cttcctggtc tggaaacctg g 21 77 20 DNA Homo sapiens 77 ctcttccctggggccctggc 20 78 22 DNA Homo sapiens 78 tgcgggggag cttgtggcac ag 22 7920 DNA Homo sapiens 79 gggcatccgc ttcctgcaga 20 80 20 DNA Homo sapiens80 ctgggcccct accctaaccc 20 81 22 DNA Homo sapiens 81 tcctggagcccgaactgtgt gt 22 82 22 DNA Homo sapiens 82 tgtcctgccc actcctcagc ct 2283 22 DNA Homo sapiens 83 ccccaggacc ccagctgtcc aa 22 84 20 DNA Homosapiens 84 aggctgacca ctgtccctct 20 85 21 DNA Homo sapiens 85 gctggcagtggcctgtgtgg a 21 86 24 DNA Homo sapiens 86 aacagtgacc aaaatgacag tgac 2487 22 DNA Homo sapiens 87 ctgcagcagt ggaaccttaa tg 22 88 22 DNA Homosapiens 88 gttcgagtgc tccagcttct tg 22 89 22 DNA Homo sapiens 89agggcatcat gctgagctac at 22 90 21 DNA Homo sapiens 90 tttagccagtggtggggttc a 21 91 20 DNA Homo sapiens 91 gttcagcagg gtggcaacat 20 92 21DNA Homo sapiens 92 gccagatggt tttcaacgac a 21 93 28 DNA Homo sapiensmisc_difference (9) Base change made to create a restriction enzymesite. 93 cagtggaagc ttaatgccca ggatgatc 28 94 35 DNA Homo sapiensmisc_difference (7)..(8) Base changes made to create a restrictionenzyme site. 94 caggaggatc cagtttagcc agtggtgggg gttca 35 95 9 DNA Homosapiens 95 ccacacccg 9 96 20 DNA Homo sapiens 96 tcagacccgg gtgagttagg20 97 20 DNA Homo sapiens 97 caatcaccag gaaaaatccc 20 98 20 DNA Homosapiens 98 ggatattcag gtaggacctg 20 99 14 DNA Homo sapiens 99 ttcctttaagaggt 14 100 10 DNA Homo sapiens 100 ttccccatga 10 101 22 DNA Homosapiens 101 ctgcagcagt ggaaccttaa tg 22 102 22 DNA Homo sapiens 102gttcgagtgc tccagcttct tg 22 103 21 DNA Homo sapiens 103 gggcatcatgctgagctaca t 21 104 21 DNA Homo sapiens 104 tttagccagt ggtggggttc a 21105 20 DNA Homo sapiens 105 gttcagcagg gtggcaacat 20 106 21 DNA Homosapiens 106 gccagatggt tttcaacgac a 21 107 26 PRT Homo sapiens 107 PheLeu Ile Val Leu Val Cys Leu Ile Phe Ser Val Leu Ser Thr Ile 1 5 10 15Glu Gln Tyr Ala Ala Leu Ala Thr Gly Thr 20 25 108 61 PRT Homo sapiens108 Leu Phe Trp Met Glu Ile Val Leu Val Val Phe Phe Gly Thr Glu Tyr 1 510 15 Val Val Arg Leu Trp Ser Ala Gly Cys Arg Ser Lys Tyr Val Gly Leu 2025 30 Trp Gly Arg Leu Arg Phe Ala Arg Lys Pro Ile Ser Ile Ile Asp Leu 3540 45 Ile Val Val Val Ala Ser Met Val Val Leu Cys Val Gly 50 55 60 109137 PRT Homo sapiens 109 Ser Lys Gly Gln Val Phe Ala Thr Ser Ala Ile ArgGly Ile Arg Phe 1 5 10 15 Leu Gln Ile Leu Arg Met Leu His Val Asp ArgGln Gly Gly Thr Trp 20 25 30 Arg Leu Leu Gly Ser Val Val Phe Ile His ArgGln Glu Leu Ile Thr 35 40 45 Thr Leu Tyr Ile Gly Phe Leu Gly Leu Ile PheSer Ser Tyr Phe Val 50 55 60 Tyr Leu Ala Glu Lys Asp Ala Val Asn Glu SerGly Arg Val Glu Phe 65 70 75 80 Gly Ser Tyr Ala Asp Ala Leu Trp Trp GlyVal Val Thr Val Thr Thr 85 90 95 Ile Gly Tyr Gly Asp Lys Val Pro Gln ThrTrp Val Gly Lys Thr Ile 100 105 110 Ala Ser Cys Phe Ser Val Phe Ala IleSer Phe Phe Ala Leu Pro Ala 115 120 125 Gly Ile Leu Gly Ser Gly Phe AlaLeu 130 135 110 66 PRT Drosophila melanogaster 110 Ile Leu Leu Ser IleVal Ile Phe Cys Leu Glu Thr Leu Pro Glu Phe 1 5 10 15 Lys His Tyr LysVal Phe Asn Thr Thr Thr Asn Gly Thr Lys Ile Glu 20 25 30 Glu Asp Glu ValPro Asp Ile Thr Asp Pro Phe Phe Leu Ile Glu Thr 35 40 45 Leu Cys Ile IleTrp Phe Thr Phe Glu Leu Thr Val Arg Phe Leu Ala 50 55 60 Cys Pro 65 111123 PRT Drosophila melanogaster 111 Asn Lys Leu Asn Phe Cys Arg Asp ValMet Asn Val Ile Asp Ile Ile 1 5 10 15 Ala Ile Ile Pro Tyr Phe Ile ThrLeu Ala Thr Val Val Ala Glu Glu 20 25 30 Glu Asp Thr Leu Asn Leu Pro LysAla Pro Val Ser Pro Gln Asp Lys 35 40 45 Ser Ser Asn Gln Ala Met Ser LeuAla Ile Leu Arg Val Ile Arg Leu 50 55 60 Val Arg Val Phe Arg Ile Phe LysLeu Ser Arg His Ser Lys Gly Leu 65 70 75 80 Gln Ile Leu Gly Arg Thr LeuLys Ala Ser Met Arg Glu Leu Gly Leu 85 90 95 Leu Ile Phe Phe Leu Phe IleGly Val Val Leu Phe Ser Ser Ala Val 100 105 110 Tyr Phe Ala Glu Ala GlySer Glu Asn Ser Phe 115 120 112 58 PRT Drosophila melanogaster 112 PheLys Ser Ile Pro Asp Ala Phe Trp Trp Ala Val Val Thr Met Thr 1 5 10 15Thr Val Gly Tyr Gly Asp Met Thr Pro Val Gly Phe Trp Gly Lys Ile 20 25 30Val Gly Ser Leu Cys Val Val Ala Gly Val Leu Thr Ile Ala Leu Pro 35 40 45Val Pro Val Ile Val Ser Asn Phe Asn Tyr 50 55 113 376 PRT Xenopus laevis113 Met Asn Glu Asn Ala Ile Asn Ser Leu Tyr Glu Ala Ile Pro Leu Pro 1 510 15 Gln Asp Gly Ser Ser Asn Gly Gln Arg Gln Glu Asp Arg Gln Ala Asn 2025 30 Ser Phe Glu Leu Lys Arg Glu Thr Leu Val Ala Thr Asp Pro Pro Arg 3540 45 Pro Thr Ile Asn Leu Asp Pro Arg Val Ser Ile Tyr Ser Gly Arg Arg 5055 60 Pro Leu Phe Ser Arg Thr Asn Ile Gln Gly Arg Val Tyr Asn Phe Leu 6570 75 80 Glu Arg Pro Thr Gly Trp Lys Cys Phe Val Tyr His Phe Thr Val Phe85 90 95 Leu Ile Val Leu Ile Cys Leu Ile Phe Ser Val Leu Ser Thr Ile Gln100 105 110 Gln Tyr Asn Asn Leu Ala Thr Glu Thr Leu Phe Trp Met Glu IleVal 115 120 125 Leu Val Val Phe Phe Gly Ala Glu Tyr Val Val Arg Leu TrpSer Ala 130 135 140 Gly Cys Arg Ser Lys Tyr Val Gly Val Trp Gly Arg LeuArg Phe Ala 145 150 155 160 Arg Lys Pro Ile Ser Val Ile Asp Leu Ile ValVal Val Ala Ser Val 165 170 175 Ile Val Leu Cys Val Gly Ser Asn Gly GlnVal Phe Ala Thr Ser Ala 180 185 190 Ile Arg Gly Ile Arg Phe Leu Gln IleLeu Arg Met Leu His Val Asp 195 200 205 Arg Gln Gly Gly Thr Trp Arg LeuLeu Gly Ser Val Val Phe Ile His 210 215 220 Arg Gln Glu Leu Ile Thr ThrLeu Tyr Ile Gly Phe Leu Gly Leu Ile 225 230 235 240 Phe Ser Ser Tyr PheVal Tyr Leu Ala Glu Lys Asp Ala Ile Asp Ser 245 250 255 Ser Gly Glu TyrGln Phe Gly Ser Tyr Ala Asp Ala Leu Trp Trp Gly 260 265 270 Val Val ThrVal Thr Thr Ile Gly Tyr Gly Asp Lys Val Pro Gln Thr 275 280 285 Trp IleGly Lys Thr Ile Ala Ser Cys Phe Ser Val Phe Ala Ile Ser 290 295 300 PhePhe Ala Leu Pro Ala Gly Ile Leu Gly Ser Gly Phe Ala Leu Lys 305 310 315320 Val Gln Gln Lys Gln Arg Gln Lys His Phe Asn Arg Gln Ile Pro Ala 325330 335 Ala Ala Ser Leu Ile Gln Thr Ala Trp Arg Cys Tyr Ala Ala Glu Asn340 345 350 Pro Asp Ser Ala Thr Trp Lys Ile Tyr Ile Arg Lys Gln Ser ArgAsn 355 360 365 His His Ile Met Ser Pro Ser Pro 370 375 114 570 PRT Homosapiens 114 Gln Gly Arg Val Tyr Asn Phe Leu Glu Arg Pro Thr Gly Trp LysCys 1 5 10 15 Phe Val Tyr His Phe Ala Val Phe Leu Ile Val Leu Val CysLeu Ile 20 25 30 Phe Ser Val Leu Ser Thr Ile Glu Gln Tyr Ala Ala Leu AlaThr Gly 35 40 45 Thr Leu Phe Trp Met Glu Ile Val Leu Val Val Phe Phe GlyThr Glu 50 55 60 Tyr Val Val Arg Leu Trp Ser Ala Gly Cys Arg Ser Lys TyrVal Gly 65 70 75 80 Leu Trp Gly Arg Leu Arg Phe Ala Arg Lys Pro Ile SerIle Ile Asp 85 90 95 Leu Ile Val Val Val Ala Ser Met Val Val Leu Cys ValGly Ser Lys 100 105 110 Gly Gln Val Phe Ala Thr Ser Ala Ile Arg Gly IleArg Phe Leu Gln 115 120 125 Ile Leu Arg Met Leu His Val Asp Arg Gln GlyGly Thr Trp Arg Leu 130 135 140 Leu Gly Ser Val Val Phe Ile His Arg GlnGlu Leu Ile Thr Thr Leu 145 150 155 160 Tyr Ile Gly Phe Leu Gly Leu IlePhe Ser Ser Tyr Phe Val Tyr Leu 165 170 175 Ala Glu Lys Asp Ala Val AsnGlu Ser Gly Arg Val Glu Phe Gly Ser 180 185 190 Tyr Ala Asp Ala Leu TrpTrp Gly Val Val Thr Val Thr Thr Ile Gly 195 200 205 Tyr Gly Asp Lys ValPro Gln Thr Trp Val Gly Lys Thr Ile Ala Ser 210 215 220 Cys Phe Ser ValPhe Ala Ile Ser Phe Phe Ala Leu Pro Ala Gly Ile 225 230 235 240 Leu GlySer Gly Phe Ala Leu Lys Val Gln Gln Lys Gln Arg Gln Lys 245 250 255 HisPhe Asn Arg Gln Ile Pro Ala Ala Ala Ser Leu Ile Gln Thr Ala 260 265 270Trp Arg Cys Tyr Ala Ala Glu Asn Pro Asp Ser Ser Thr Trp Lys Ile 275 280285 Tyr Ile Arg Lys Ala Pro Arg Ser His Thr Leu Leu Ser Pro Ser Pro 290295 300 Lys Pro Lys Lys Ser Val Val Val Lys Lys Lys Lys Phe Lys Leu Asp305 310 315 320 Lys Asp Asn Gly Val Thr Pro Gly Glu Lys Met Leu Thr ValPro His 325 330 335 Ile Thr Cys Asp Pro Pro Glu Glu Arg Arg Leu Asp HisPhe Ser Val 340 345 350 Asp Gly Tyr Asp Ser Ser Val Arg Lys Ser Pro ThrLeu Leu Glu Val 355 360 365 Ser Met Pro His Phe Met Arg Thr Asn Ser PheAla Glu Asp Leu Asp 370 375 380 Leu Glu Gly Glu Thr Leu Leu Thr Pro IleThr His Ile Ser Gln Leu 385 390 395 400 Arg Glu His His Arg Ala Thr IleLys Val Ile Arg Arg Met Gln Tyr 405 410 415 Phe Val Ala Lys Lys Lys PheGln Gln Ala Arg Lys Pro Tyr Asp Val 420 425 430 Arg Asp Val Ile Glu GlnTyr Ser Gln Gly His Leu Asn Leu Met Arg 435 440 445 Val Ile Lys Glu LeuGln Arg Arg Leu Asp Gln Ser Ile Gly Lys Pro 450 455 460 Ser Leu Phe IleSer Val Ser Glu Lys Ser Lys Asp Arg Gly Ser Asn 465 470 475 480 Thr IleGly Ala Arg Leu Asn Arg Val Glu Asp Lys Val Thr Gln Leu 485 490 495 AspGln Arg Leu Ala Leu Ile Thr Asp Met Leu His Gln Leu Leu Ser 500 505 510Leu His Gly Gly Ser Thr Pro Gly Ser Gly Gly Pro Pro Arg Glu Gly 515 520525 Gly Ala His Ile Thr Gln Pro Cys Gly Ser Gly Gly Ser Val Asp Pro 530535 540 Glu Leu Phe Leu Pro Ser Asn Thr Leu Pro Thr Tyr Glu Gln Leu Thr545 550 555 560 Val Pro Arg Arg Gly Pro Asp Glu Gly Ser 565 570

What is claimed is:
 1. A method for diagnosing the presence of apolymorphism in human KCNE1 (the coding region of which is bases 193-579of SEQ ID NO:3) which causes long QT syndrome wherein said method isperformed by means which identify the presence of said polymorphism,wherein said polymorphism is one which results in the presence of aKCNE1 polypeptide of SEQ ID NO:4 with an altered amino acid, saidaltered amino acid being selected from the group consisting of: a) a Leuat residue
 74. 2. The method of claim 1 wherein said polymorphism isselected from the group consisting of: a) a T at base 413 of SEQ IDNO:3.
 3. The method of claim 1 wherein said means comprises using asingle-stranded conformation polymorphism technique to assay for saidpolymorphism.
 4. The method of claim 1 wherein said means comprisessequencing human KCNE1.
 5. The method of claim 1 wherein said meanscomprises performing an RNAse assay.
 6. The method of claim 2 whereinsaid means comprises using a single-stranded conformation polymorphismtechnique to assay for said polymorphism.
 7. The method of claim 2wherein said means comprises sequencing human KCNE1.
 8. The method ofclaim 2 wherein said means comprises performing an RNAse assay.
 9. Amethod for diagnosing long QT syndrome in a person wherein said methodcomprises sequencing a KCNE1 polypeptide from said person or sequencingKCNE1 polypeptide synthesized from nucleic acid derived from said personwherein the presence of: a) a Leu at residue 74, is indicative of longQT syndrome.
 10. A method for diagnosing a polymorphism which causeslong QT syndrome comprising determining the KCNE1 sequence in a patientby preparing cDNA from RNA taken from the patient, and sequencing saidcDNA to determine the presence or absence of the polymorphism consistingof a T at base 413 of SEQ ID NO:3.
 11. A method of assessing a risk in ahuman subject for long QT syndrome which comprises screening saidsubject for a polymorphism in a KCNE1 gene by comparing the sequence ofthe KCNE1 gene or its expression products isolated from a tissue sampleof said subject with a wild-type KCNE1 gene or its expression products,wherein a polymorphism consisting of a T at base 413 of SEQ ID NO:3 or aLeu at residue 74 of SEQ ID NO:4 is indicative of a risk for long QTsyndrome.
 12. The method of claim 11 wherein said expression product isselected from the group consisting of mRNA of the KCNE1 gene (SEQ IDNO:3) and a KCNE1 polypeptide (SEQ ID NO:4) encoded by the KCNE1 gene.13. The method of claim 11 wherein one or more of the followingprocedures is carried out: (a) observing shifts in electrophoreticmobility of single-stranded DNA from said sample on non-denaturingpolyacrylamide gels; (b) hybridizing a KCNE1 gene probe to genomic DNAisolated from said sample under conditions suitable for hybridization ofsaid probe to said gene; (c) determining hybridization of anallele-specific probe to genomic DNA from said sample; (d) amplifyingall or part of the KCNE1 gene from said sample to produce an amplifiedsequence and sequencing the amplified sequence; (e) determining bynucleic acid amplification the presence of a specific KCNE1 polymorphicallele in said sample; (f) molecularly cloning all or part of the KCNE1gene from said sample to produce a cloned sequence and sequencing thecloned sequence; (g) determining whether there is a mismatch betweenmolecules (1) KCNE1 gene genomic DNA or KCNE1 mRNA isolated from saidsample, and (2) a nucleic acid probe complementary to the humanwild-type KCNE1 gene DNA, when molecules (1) and (2) are hybridized toeach other to form a duplex; (h) amplification of KCNE1 gene sequencesin said sample and hybridization of the amplified sequences to nucleicacid probes which comprise wild-type KCNE1 gene sequences; (i) screeningfor a deletion mutation; (j) screening for a point mutation; (k)screening for an insertion mutation; (l) determining in situhybridization of the KCNE1 gene in said sample with one or more nucleicacid probes which comprise the KCNE1 gene sequence or the polymorphicKCNE1 gene sequence; (m) immunoblotting; (n) immunocytochemistry; (o)assaying for binding interactions between KCNE1 gene protein isolatedfrom said sample and a binding partner capable of specifically bindingthe polypeptide expression product of a KCNE1 polymorphic allele and/ora binding partner for the KCNE1 polypeptide having the amino acidsequence set forth in SEQ ID NO:4; and (p) assaying for the inhibitionof biochemical activity of said binding partner.